2612
Rabia Raza
Yu Bai
Huwei Liu
Beijing National Laboratory for
Molecular Sciences, Key
Laboratory of Bioorganic
Chemistry and Molecular
Engineering of Ministry of
Education, Institute of Analytical
Chemistry, College of Chemistry
and Molecular Engineering,
Peking University, Beijing,
P. R. China
Received February 24, 2018
Revised May 1, 2018
Accepted May 3, 2018
Electrophoresis 2018, 39, 2612–2618
Research Article
Development of a fast CE method for high
throughput screening of ecto-5 -nucleotidase inhibitors
Overexpression of ecto-5 -nucleotidase is found to be linked to cancer progression and
other diseases which makes screening of its inhibitors on demand. Unfortunately, there
is scarcity of reported ecto-5 -nucleotidase inhibitors because of unavailability of simple,
fast and efficient methods to study its inhibition. In this study, we have developed a
very fast, reverse polarity capillary electrophoresis based method for screening of ecto-5 nucleotidase inhibitors. Both EOF and electrophoretic mobility were maintained in same
direction by dynamically coating lining of capillary and applying negative voltage, resulting
in appearance of both adenosine monophosphate (AMP) and adenosine peaks in less than
2 min. Separations were performed in reverse polarity using -20 kV. Hexadimethrine
bromide (HDB) was used as a buffer additive (0.025% in 40 mM borate buffer). An online
sample stacking was attained by a special sweeping effect which enabled a long (65 s)
injection time without compromise in peak shape. Using this newly developed method,
both AMP and adenosine peaks were obtained with a baseline resolution, in less than
two minutes. Method was validated by measuring km , and Vmax values of human ecto5 -nucleotidas and Ki value of standard inhibitor quercitin against this enzyme. Method
is simple and fast and can be conveniently optimized for high throughput screening of
ecto-5 -nucleotidase inhibitors.
Keywords:
Capillary electrophoresis / Ecto-5 -nucleotidase / High throughput screening /
Inhibition
DOI 10.1002/elps.201800105
1 Introduction
Ecto-5’-nucleotidase (CD73) (EC 3.1.3.5) is a membrane
bound ectoenzyme, involved in production of extracellular
adenosine by catalyzing hydrolysis of ribo and deoxyribonucleotide 5’-monophosphates to their corresponding nucleosides. It hydrolyzes AMP to adenosine and hence serves as
the major control point for extracellular adenosine levels [1].
Over expression of ecto-5’-nucleotidase leads to enhanced
concentration of adenosine in extracellular environment
which affects a number of biological processes. Variations in
adenosine receptors, adenosine membrane transporters, and
Correspondence: Professor Huwei Liu, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry and Molecular
Engineering, Peking University, Beijing 100871, P. R. China
E-mail: hwliu@pku.edu.cn
Abbreviations: ADP, Adenosine 5 -diphosphate; AMP,
Adenosine 5 -monophosphate; ATP, Adenosine 5 triphosphate; cAMP, Cyclic adenosine 5 -monophosphate;
DAD, Diode array detector; HDB, Hexadimethrine bromide
C 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
corresponding intracellular signaling networks are also reported to be associated with development of preeclampsia [2, 3]. Ecto-5 -nucleotidase is reported to be over expressed on many cancer types including thyroid, ovarian,
breast, prostate, leukemia, bladder, gastric, eosophageal, and
colon cancer [4–7]. It is also proposed to be used as a
diagnostic tool for cancer [8]. Thus, inhibition of ecto-5 nucleotidase is an important field of cancer research and
other related physiological functions [9]. Regardless of its
sheer medical significance, inhibition of ecto-5 -nucleotidase
is a relatively less studied area due to scarcity of fast
and efficient methods for high throughput analysis of its
inhibitors.
Various types of enzyme assays using different analytical
techniques are being employed to study ecto-5 -nucleotidase.
Every assay type has its own pros and cons. The most common type of assays are spectrophotometric assays [10]. These
assays usually measure inorganic phosphate, released by hydrolysis of AMP through their reaction with a colouring agent.
They are prone to low sensitivity and false positive signals
due to ubiquitous presence of inorganic phosphates in water
Color Online: See the article online to view Figs. 2 and 4 in color.
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Electrophoresis 2018, 39, 2612–2618
and buffers. Also chromophores and inhibitors, containing
phosphate, cannot be precisely determined by these assays.
High sensitivity can be achieved by using radiometric assays [11] but they require expensive radioactive reagents and
scintillation counters which limit their applicability. Chromatographic assays [12] are also an alternative but they require relatively large amounts of solvents and reagents and
are usually time consuming and are not appropriate for high
throughput screening of large libraries of inhibitors. Another
type of assays are luminescent assays [13]. These assays require presence of a luciferase coupling enzyme and often
require a large amount of enzyme. Almost all of the above
enzyme assays are limited to water soluble inhibitors owing
to their incompatibility with other solvents. So an alternative, fast and efficient method is highly required for study of
ecto-5 -nucleotidase inhibitors.
Capillary electrophoresis (CE) has become a routine analytical technique for analysis of both small and large analytes, owing to its several advantages like good separation
efficiency, high sensitivity, diverse detection modes, fast analysis and extreme small sample consumption [14, 15]. It has
also been extensively employed in enzymatic studies including enzyme assays, enzyme kinetics and enzyme inhibition [16–19]. Another advantage of CE based assays is their
suitability for compounds containing chromophores or phosphates that otherwise cannot be tested by spectrophotometric
methods [20].
Zhu et al. [21] reported a CE based method for adenine
nucleotides analysis in Saccharomyces cerevisiae in which ATP,
ADP, AMP, and cAMP were separated using a non-coated
capillary. Although method was efficient in separation of
ATP and its metabolites but it cannot be used for ecto-5 nucleotidase study as the separation time for AMP was 30 min
that is too high to be used in a high throughput screening. Recently a capillary zone electrophoresis method has
been reported for simultaneous determination of intracellular nucleotides and coenzymes in Yarrowia lipolytica [22]. The
method is based on comparison of separation efficiency of
bare fused silica capillary and sol–gel coated capillary containing porphyrin-brucine conjugate. Although, the method efficiently separated many nucleosides and nucleotides but it is
also inappropriate for ecto-5 -nucleotidase study as long separation time for AMP and adenosine. Iqbal et al. [23] reported
off-capillary and in-capillary CE based methods for membrane bound ecto-5 -nucleotidase. In-capillary enzymatic reaction required concentrated solutions of enzyme and substrate and may not be so efficient for low activity enzyme. In
off-capillary method, separation of substrates and products
was achieved in eight min using bare fused-silica capillary
and applying positive voltage. As electrophoretic mobility of
AMP (anion) was towards anode (opposite to EOF) in normal
polarity separation, so it took eight min to elute along with
EOF. While adenosine (product) being neutral molecule, was
also eluted along with EOF but earlier than AMP. So development of more efficient and faster CE based method was still
desired to be opted for high throughput screening of ecto-5 nucleotidase inhibitors. In our work we have used the idea
C 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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to reverse the EOF to get faster separation of product and
substrate than the previous methods.
2 Materials and methods
2.1 Reagents and chemicals
All the reagents used in experimental work were of analytical grade and were used without any additional purification. Adenosine 5 -monophosphate, adenosine, quercitin,
pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), human recombinant ecto-5 nucleotidases, and HDB were purchased from Sigma Aldrich
(China). All other laboratory reagents like NaOH, MgCl2 ,
CaCl2 , Tris HCl, sodium tetraborate, Tris base, and NaCl
were purchased from Xilong Chemical Company (Guangdong, China). Dimethyl sulfoxide (DMSO), hydrochloric acid
and sodium hydroxide were purchased from J&K Scientific
(Beijing, China). Deionized purified water was obtained from
Hangzhou Wahaha Group (Zhejiang, China).
2.2 Instrumental conditions for CE (optimized
method)
An Agilent 7100 CE system equipped with a UV detector
which was further coupled with a DAD detector was used (Agilent Technologies, Palo Alto, CA, USA). Agilent ChemStation software was used for data assembly and peak area study.
The sample storing unit and capillary temperature were set
at 25°C for analysis. A fused silica capillary having inner and
outer diameter 50 and 360 ␮m respectively, was purchased
from Sino Sumtech (Handan. Hebei, China). Total capillary
length was 35 cm among which 26.5 cm was effective length.
A 40 mM borate buffer with 0.025% HDB and pH 8 was
used as background electrolyte (BGE). The separation voltage
was optimized at 20 kV in reverse polarity. Detection of AMP
and adenosine was accomplished with help of direct UV absorbance at 260 nm. Samples were injected hydrodynamically
for 65 s. New capillary was flushed with 0.1 N NaOH, H2 O
and BGE successively for 20 min each. During the analysis,
the capillary was conditioned by rinsing with 0.1 N NaOH for
2 min and with water and BGE for 1 min after each sample.
2.3 Validation of method
Initial validation of developed method was performed by
quantitative determination of adenosine. For this purpose,
adenosine was dissolved in assay buffer to get a stock solution of 5 mM. Serial dilutions from 0.1 to 5000 ␮M were
prepared from the stock solution and equal amount of heat
inactivated ecto-5 -nucleotidase was added to all dilutions to
provide maximum similarity with original assay conditions.
A standard calibration curve was obtained and LOQ and LOD
of the method were calculated.
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2.4 Determination of Km and Vmax values of human
ecto-5 -nucleotidase
To determine the Km and Vmax values of human ecto-5 nucleotidase, enzymatic reaction was performed outside capillary using the reported assay protocol [24]. Different concentrations of substrate (AMP) ranging from 6.25 to 500 ␮M
were prepared in assay buffer (10 mM tris HCl, 1mM CaCl2 ,
2 mM MgCl2 and pH 7.4). 10 ␮L human ecto-5 -nucleotidase
(1.2 ␮g/mL) was incubated with different concentrations of
substrate at 37°C for 10 min, in the presence of assay buffer.
Total assay volume was kept 100 ␮L. To stop the enzymatic
reaction, reaction mixtures were heated at 99°C for 20 min
and 20 ␮L of each reaction mixture was shifted to CE vials to
be injected into CE. The separation was performed using the
optimized method. The area of product peak was calculated as
percentage of total area (substrate + product area) at 260 nm.
As substrate AMP and product adenosine were chemically
related and showed maximum UV absorption at same wavelength of quantification (260 nm), any external factor influenced peak area of both. However use of an internal standard
can further eliminate any injection volume related imprecision. Data were fitted to the Michaelis-Menten equation using
the PRISM 5.0 (GraphPad, San Diego, CA) software.
2.5 Determination of ecto-5 -nucleotidase inhibition
To evaluate the efficiency of method for inhibition studies,
two standard inhibitors of ecto-5 -nucleotidase (quercitin and
PPADS) were tested. Stock solutions of quercitin and PPADS
(10 mM) were prepared in 10% DMSO and their inhibition
potencies were tested against ecto-5 -nucleotidase. Quercitin
was further analyzed by making its three fold serial dilutions
in assay buffer. The assay mixture contained 10 ␮L of test
compound, 10 ␮L of human ecto-5 -nucleotidase (1.2 ␮g/mL)
and 70 ␮L of assay buffer. The buffer comprised of MgCl2
(2 mM), Tris HCl (10 mM), CaCl2 (1 mM), at pH 7.4 and
was allowed to preincubate at 37°C for 10 min. Subsequently,
addition of substrate i.e. adenosine monophosphate (10 ␮L)
at a final concentration of 500 ␮M resulted in initiation of enzymatic reaction and allowed to proceed for 10 min at 37°C.
Enzymatic reaction was ceased by providing 99°C temperature for 20 min and then the reaction mixture was subjected
to analysis in CE. Inhibition percentage was calculated by
comparing the product formed in the sample and in the control. All experiments were performed two times in triplicate.
Inhibition Constant (Ki ) value was calculated using PRISM
5.0 (GraphPad, San Diego, CA) software.
Electrophoresis 2018, 39, 2612–2618
somehow limited in terms of sensitivity. In detection cell,
optical path length is quite short leading to a low detectable
concentration in CE coupled with UV absorption detector
with normal detection range 1–10 ␮M which is lower than
HPLC. However, operational modifications can be used to
enhance sensitivity and performance considerably.
The present study focused on the development of a CE
based method using dynamically coated capillary for efficient and fast separation of enzymatic products of ecto-5 nucleotidase. Adenosine monophosphate was used as the
substrate of enzyme and a successful separation of both AMP
(negatively charged) and adenosine (neutral) molecules was
attained in less than 2 min. Different operational parameters
of the method were optimized to attain best separation in
minimum time period.
3.1.1 Optimization of BGE
Choice of an appropriate BGE is a prerequisite to develop
an efficient CE method. In order to find the most efficient
CE separation BGE, phosphate, borate and Tris buffers were
tested in initial experiments (data not shown). Each buffer
was evaluated for its buffering capacity, current production
in capillary under applied voltage and most importantly for
separation efficacy. Borate was found to be the best among
all the three buffers for its separation efficiency and low Joule
heat production. Next step was to find the most appropriate
BGE concentration and pH. For determination of optimum
buffer strength, a range of borate buffer concentrations were
examined. Five solutions of borate buffer ranging from 10
to 100 mM borate were tested for their buffering capacity
and efficiency in separation of AMP and adenosine. It was
observed that increasing the concentration of BGE resulted in
improvement of separation and quantification efficiency but
at the same time it also enhanced the Joule heat and migration
time. A borate buffer with 40 mM concentration was found
to be the most appropriate in separation of products with
60 ␮A current without compromising migration time. The
pH plays a vital role in electrophoretic separations. To find
out the most optimum pH for the best separation of substrate
and products, solutions of borate buffers having a range of pH
from 7 to 11 were prepared and checked for their separation
efficiency. Borate buffers with pH 7–9 were prepared using
sodium tetraborate and hydrochloric acid while buffers with
pH 9–11 were prepared using sodium tetraborate and sodium
hydroxide solutions. It was found that the developed method
worked efficiently in pH range 7.5 to 8.5. So a 40 mM borate
buffer with pH 8 was used in subsequent experiments.
3 Results and discussion
3.1 CE method optimization for determination and
quantification of enzymatic products of
ecto-5 -nucleotidase
CE has emerged as a cost effective, fast and efficient separation technique and is being used extensively for enzymatic
studies in recent years [25]. However, its applications are
C 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.1.2 Optimization of BGE additive
The use of dynamic coating of capillaries is preferred over
permanent coatings owing to its benefits like easy to generate, use of cheap fused silica capillary and compatibility with
a wide range of buffer concentration. A polycationic buffer
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3.1.3 Optimization of separation voltage
Separation voltage is a very important parameter involved in
electrophoretic separations because all its mechanism is controlled by amount of voltage applied. A negative voltage was
used in all experiments. To find out the most optimum voltage to get best value of current, Joule heating and separation
time, negative voltages in range of 5 to 25 kV were tested. Increasing voltage resulted in shorter migration time but higher
current and relatively low resolution. Among different voltage values, -20 kV was found to be the most appropriate for
providing a baseline resolution of AMP and adenosine in
less than two min without crossing maximum current limit
(100 ␮A).
Effect of injection time on peak area
300
Peak area of adenosin
additive “HDB” was used to coat the capillary dynamically.
Appropriate amount of HDB was very critical to alter the behavior of EOF. The effect of amount of HDB in BGE was studied by using varying concentrations of HDB (0.1–0.001%). It
was observed that increasing HDB concentration resulted in
enhanced sensitivity and improvement in peak shape upto
0.025% HDB in BGE. After this concentration, there was no
considerable effect on separation efficacy and sensitivity.
This buffer additive played a key role in controlling the
whole mechanism of separation in this method. Due to presence of cationic additive HDB, EOF was reversed i.e. from
anode to cathode while electrophoretic mobility of negatively
charged AMP was also towards anode. Negatively charged
substrate (AMP) moved towards anode under the influence
of electric charge while the neutral product ions (adenosine) moved along with EOF, towards anode. Movement of
charged ions and EOF in the same direction resulted in appearance of both AMP and adenosine peaks in a very short
time.
Role of HDB was not limited to short migration time
only. It also served as a tool for online sample concentration by offering a special sweeping effect. Although the bulk
flow of the BGE was toward anode under influence of EOF,
but positively charged HDB ions were being attracted toward
cathode. This opposite movement of HDB ions made a temporary barrier against the movement of sample ions (AMP
& Adenosin). The result of this barrier was concentration of
sample ions in a narrow band and offered an online stacking phenomenon which served to improve sensitivity of the
method.
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240.7
250
211.4
200
173.1
150
94.6
100
50
17.6
0
5
25
45
60
65
Injection time (s)
Figure 1. Effect of injection time on peak area of adenosine using
-20 kV, 40 mM borate (0.025% HDB), 35 cm (effective 26.5 cm)
capillary length.
with increasing injection duration. Maximum sensitivity was
achieved at 65-s injection time without compromising peak
shape. This specialty of long injection time (65 s) is again attributed to the stacking effect of HDB that allowed an online
sample concentration and resulted in a narrow but concentrated sample band. Effect of injection time on peak area of
adenosine is depicted in Fig. 1). Injections longer than 65 s
resulted in tailed unsymmetrical peaks.
3.1.5 Optimization of capillary length
Initially both permanently coated and uncoated capillaries
were tested for the experiment and bare fused silica capillary with manual dynamic coating was found better so in
rest of experiments, this capillary was used. Capillary length
is an important factor which mainly influence on migration
time of the analytes. Longer capillaries provide better separations and less Joule heat but also increase migration times.
Different capillary lengths were tested to get minimum migration time within less than 100 ␮A current limit. A capillary with 35 cm total length (26.5 cm effective length) was
found to be the best to overcome the Joule heating problems and delayed migration times. Figure 2 shows separation of AMP and adenosine using optimized CE conditions which were able to separate AMP and adenosine in
less than 2 min without compromising resolution and peak
shape.
3.1.4 Optimization of sample injection time
3.1.6 Quantitative determination of adenosine
Long Injections in electrophoretic operations can load more
sample to increase sensitivity but in practice usually short
injection times (2–3 s) are used because long injections also
result in tailed and skewed peaks. Effect of different hydrodynamic injections times (5, 25, 45, 60, 65, 75 s) on sensitivity and peak shape was observed using a constant −20 kV
voltage. The peak areas of AMP and adenosine were increased
C 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
After optimizing all the separation conditions, the method
was validated and enzymatic product adenosine was quantified. The value of determination coefficient (R2 ) was 0.9999
using adenosine concentration from 0.1 to 5000 ␮M. The
determined LOD and LOQ were 1.76 ± 0.26 ␮M and
535 ± 0.30 ␮M, respectively (Table 1).
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Electrophoresis 2018, 39, 2612–2618
Figure
2. Separation
of
adenosine monophosphate
(AMP) and adenosine (Ad)
using 40 mM borate buffer
with HDB 0.025%, −20 kV
voltage (reverse polarity),
65 s hydrodynamic Injection,
35 cm capillary (effective
length = 26.5 cm).
Table 1. LOD, LOQ, linearity of calibration curve and migration
time values of adenosine by newly developed CE based
method
LOD ± SD (␮M)
LOQ ± SD (␮M)
Linearity of calibration curve (R2 value)
Migration time ± SD (mean value) (min)
%RSD of migration time (min)
1.76 ± 0.26
5.35 ± 0.30
0.9999
1.46 ± 0.01
0.004
Note: SD: Standard deviation; RSD: Relative standard deviation.
3.2 Application of newly developed CE method for
study of ecto-5 -nucleotidase
To evaluate the applicability of the CE-based method, it
was used for study of enzymatic reaction of human ecto5 -nucleotidase using AMP as substrate. Enzymatic assay was performed outside capillary and the enzymatic
reaction mixture was then transferred to CE vials for
quantification.
Figure 3. Michaelis-Menten plots for human recombinant ecto5 -nucleotidase.
3.2.1 Determination of Km and Vmax values of human
ecto-5 -nucleotidase
The kinetic parameters Km and Vmax of human ecto5 -nucelotidases were determined using varying substrate
(AMP) concentrations ranging from 6.25 to 500 ␮M. Peak
area of the product (adenosine) corresponding to each concentration was used as the parameter to quantify it. Peak
height is also used some times for the same purpose but peak
area is a more accurate parameter as height is influenced by
many factors and may not represent the true amount of product formed. Data were fitted to the Michaelis–Menten equation. Michaelis–Menten plots for human ecto-5 -nucleotidase
is shown in Fig. 3.
The obtained Km value for the human ecto-5 nucleotidase was 22.6 ± 3.3 ␮M which was in very good
agreement with previously reported Km value for ecto-5 nucleotidase (23 ␮M) using a CE method [23] and also with
C 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
a spectrophotometric method (25 ␮M) [26]. The Vmax was
found to be 43.94 ± 3.6 ␮mol/min/mg protein which was
also quite close to the previously reported Vmax values that
was 32 ␮mol/min/mg of protein [23].
3.2.2 Determination of ecto-5 -nucleotidase inhibition
The newly developed method was also validated by measuring inhibition constant of standard inhibitors of ecto-5 nucleotidase. Stock solutions of standard inhibitors quercitin
and PPADS were tested initially at 1 mM final concentration
and their products were quantified using the optimized CE
method. PPADS exhibited 44% inhibition at 1 mM concentration while quercitin being more potent inhibitor exhibited
90% inhibition at given concentration. Tested inhibitors did
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Figure 4. Electropherogram showing inhibition of ecto-5 -nucleotidase in the presence of 0.1 mM quercitin and control with no quercitin.
products for high throughput screening of inhibitors, performed off capillary.
4 Concluding remarks
Figure 5. Concentration
nucleotidase by quercitin.
dependent
inhibition
of
ecto-5 -
not show absorbance at quantification wavelength (260 nm).
Figure 4 shows reduced peak area of adenosine due to
inhibition of ecto-5 -nucleotidase, in the presence of 0.1 mM
quercitin.
To evaluate inhibition limit of quercitin further, its serial
dilutions were tested and product area against corresponding
inhibitor concentration was calculated. Inhibition Constant
(Ki) value was calculated. Concentration-inhibition curve of
quercitin is shown in Fig. 5 and the Ki values of the quercitin
was found to be 0.63 ± 0.1 ␮M.
Enzymatic study by this method require only few microliters of sample, so miniaturized off capillary assays can
be performed in 96 well plates and can be analyzed in very
short analyses time by using this method. Hence the method
can be optimized and adopted for rapid analyses of reaction
C 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
In conclusion, a very fast CE based method has been successfully developed and optimized for separation and quantification of charged as well as neutral nucleotides. Method
has been successfully used for study of enzymatic reaction of
human ecto-5 -nucleotidase. Enzymatic reactions were performed outside capillary and the enzymatic products were
separated and quantified with this CE method. A polycationic
molecule HDB was used as a buffer additive in 40 mM borate
buffer (pH 8) which made a dynamic coating on inner side of
the capillary and reversed EOF. Positively charged HDB also
contributed toward in-capillary sample concentration by making a temporary barrier of positive charges in front of sample
and enabled a long (65 s) injection time resulting in narrow
and concentrated sample band. Separations were performed
using -20 kV and by using this method, both AMP (negatively
charged) and adenosine (neutral) peaks were obtained with
a baseline resolution, in less than 2 min. Adenosine being
neutral molecule was eluted along with EOF. Method can be
conveniently used for study of ecto-5 -nucleotidase and high
throughput screening of variety of inhibitors in very short
analysis time. However, those neutral molecules that exhibit
UV absorption at 260 nm wavelength, can interfere with precise quantification of adenosine. Some additional modifications like protonation of analyte may be performed prior to
analysis, for such compounds.
This work is supported by the National Natural Science Foundation of China (No. 21527809) and Foreign Youth Talent Recruitment Program.
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R. Raza et al.
The authors have declared no conflict of interest.
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