Rapid, reliable, and sensitive detection of adenosine deaminase activity by UHPLC-Q-Orbitrap HRMS and its application to inhibitory activity evaluation of traditional Chinese medicines

Article abstract of DOI:10.1016/j.jpba.2018.02.045

Adenosine deaminase (ADA), which is a key enzyme in the metabolism of purine nucleosides, plays important roles in diverse disorders, such as tuberculosis, diabetes, liver disorders, and cancer. Determination of the activities of ADA and its isoenzymes in body fluids has received considerable attention in the diagnosis and treatment of relative diseases. Ultraviolet spectroscopy with adenosine (AD) as a substrate is a classical approach for screening potential ADA inhibitors by measuring the decrease in substrate (AD) at 265 nm or increase in the product (inosine) at 248 nm. However, AD and inosine share a very close maximum absorption wavelength, and the reaction is uncertain and is frequently interfered by the background color of matrix compounds or plant extracts. Thus, the method usually yields false positive or negative results. In this study, a novel, rapid, sensitive, and accurate ultra-high-performance liquid chromatography-Q exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometric (UHPLC-Q-Orbitrap HRMS) method was developed for determining and screening ADA inhibitors by directly determining the deamination product of AD, inosine. A proper separation was achieved for inosine and chlormequat (internal standard) within 2 min via isocratic elution (0.1% formic acid:methanol = 85:15, v/v) at a flow rate of 0.3 mL min^?1 on a Waters ACQUITY HSS T3 column (2.1 mm × 100 mm, 1.8 μm) following a simple precipitation of proteins. The intra- and inter-day precisions of the developed method were below 7.17% and 8.99%, respectively. The method exhibited advantages of small total reaction volume (60 μL), short running time (2 min), high sensitivity (lowest limit of quantification of 0.02 μM for inosine), and low cost (small enzyme consumption of 0.007 unit mL^?1 for ADA and substrate of 3.74 μM for AD in individual inhibition), and no matrix effects (101.64%–107.12%). Stability results showed that all analytes were stable under the investigated conditions. The developed method was successfully applied to the detection of the inhibitory activity of ADA from traditional Chinese medicines.

Full text of DOI:10.1016/j.jpba.2018.02.045

Accepted Manuscript
Title: Rapid, reliable, and sensitive detection of adenosine
deaminase activity by UHPLC-Q-Orbitrap HRMS and its
application to inhibitory activity evaluation of traditional
Chinese medicines
Authors: Shenglan Qi, Huida Guan, Gang Deng, Tao Yang,
Xuemei Cheng, Wei Liu, Ping Liu, Changhong Wang
PII:
DOI:
Reference:
S0731-7085(17)33129-1
https://doi.org/10.1016/j.jpba.2018.02.045
PBA 11813
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date:
Revised date:
Accepted date:
20-12-2017
17-2-2018
20-2-2018
Please cite this article as: Shenglan Qi, Huida Guan, Gang Deng, Tao Yang,
Xuemei Cheng, Wei Liu, Ping Liu, Changhong Wang, Rapid, reliable, and sensitive
detection of adenosine deaminase activity by UHPLC-Q-Orbitrap HRMS and its
application to inhibitory activity evaluation of traditional Chinese medicines, Journal of
Pharmaceutical and Biomedical Analysis https://doi.org/10.1016/j.jpba.2018.02.045
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Rapid, reliable, and sensitive detection of adenosine deaminase activity by UHPLC-Q-Orbitrap HRMS
and its application to inhibitory activity evaluation of traditional Chinese medicines
a
d
c
c
Shenglan Qi a, 1, Huida Guan a, 1, Gang Deng , Tao Yang , Xuemei Cheng a, b, Wei Liu *, Ping Liu *,
Changhong Wang a, b
*
aInstitute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine; The MOE Key
Laboratory for Standardization of Chinese Medicines and The SATCM Key Laboratory for New Resources and
Quality Evaluation of Chinese Medicines, 1200 Cailun Rood, Shanghai 201203, China
bShanghai R&D Centre for Standardization of Chinese Medicines, 199 Guoshoujing Road, Shanghai 201203,
China
cKey Laboratory of Liver and Kidney Diseases (Ministry of Education), Institute of Liver Diseases, Shuguang
Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, 528 Zhangheng Road, Shanghai
201203, China
dInstitute of Cardiovascular Disease, Shuguang Hospital Affiliated with Shanghai University of Traditional
Chinese Medicine, 528 Zhangheng Road, Shanghai 201203, China
1Shenglan Qi and Huida Guan contributed equally to this work.
*Correspondence to: Professor Chang-hong Wang, Ping Liu and Wei Liu, The Institute of Chinese Materia
Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. Tel: 086-021-51322511,
Fax: 086-021-51322519, E-mail: wchcxm@hotmail.com (C.H. Wang); Key Laboratory of Liver and Kidney
Diseases (Ministry of Education), Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese
Medicine, Shanghai 201203, China. liuliver@vip.sina.com (P. Liu); Institute of Liver Diseases, Shuguang
Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
lwhzayl@163.com (W. Liu).
1

Graphical abstract
Highlights


ADA plays an important role in drug development and diseases detection
An UHPLC-Q-Orbitrap HRMS method was developed to determine ADA activity.



The assay is run within 2 min with acceptable sensitivity and repeatability.
The method is economical for substrate and enzyme consumption.
The method is successfully applied to detect inhibitive activity of herbal medicines.
Abstract
Adenosine deaminase (ADA), which is a key enzyme in the metabolism of purine nucleosides, plays
important roles in diverse disorders, such as tuberculosis, diabetes, liver disorders, and cancer. Determination of
the activities of ADA and its isoenzymes in body fluids has received considerable attention in the diagnosis and
treatment of relative diseases. Ultraviolet spectroscopy with adenosine (AD) as a substrate is a classical approach
for screening potential ADA inhibitors by measuring the decrease in substrate (AD) at 265 nm or increase in the
product (inosine) at 248 nm. However, AD and inosine share a very close maximum absorption wavelength, and
the reaction is uncertain and is frequently interfered by the background color of matrix compounds or plant
extracts. Thus, the method usually yields false positive or negative results. In this study, a novel, rapid, sensitive,
2

and accurate ultra-high-performance liquid chromatography-Q exactive hybrid quadrupole orbitrap
high-resolution accurate mass spectrometric (UHPLC-Q-Orbitrap HRMS) method was developed for
determining and screening ADA inhibitors by directly determining the deamination product of AD, inosine. A
proper separation was achieved for inosine and chlormequat (internal standard) within 2 min via isocratic elution
(0.1% formic acid:methanol = 85:15, v/v) at a flow rate of 0.3 mL·min−1 on a Waters ACQUITY HSS T3 column
(2.1 mm × 100 mm, 1.8 μm) following a simple precipitation of proteins. The intra- and inter-day precisions of
the developed method were below 7.17% and 8.99%, respectively. The method exhibited advantages of small
total reaction volume (60 μL), short running time (2 min), high sensitivity (lowest limit of quantification of 0.02
μM for inosine), and low cost (small enzyme consumption of 0.007 unit·mL−1 for ADA and substrate of 3.74 μM
for AD in individual inhibition), and no matrix effects (101.64%–107.12%). Stability results showed that all
analytes were stable under the investigated conditions. The developed method was successfully applied to the
detection of the inhibitory activity of ADA from traditional Chinese medicines.
Keywords: adenosine deaminase; inhibitor; UHPLC-Q-Orbitrap HRMS; traditional Chinese herbal medicine
3

Introduction
Adenosine deaminase (adenosine aminohydrolase, EC3.5.4.4, ADA) plays a relevant role in intracellular and
extracellular nucleotide metabolism and specifically participates in the irreversible hydrolytic deamination of
adenosine (AD) and deoxyadenosine to produce inosine and 2’-deoxyinosine, respectively, and ammonia (Fig. 1)
[1, 2]. Accumulating evidence indicates that dysfunctional ADA in human body is closely related to many
important diseases, such as tuberculosis [3], diabetes [4, 5], liver disorders [6, 7], and cancer [8-11]. Given its
significance in pathology, ADA is an important target for drug development and disease detection. Elevated ADA
levels in the sera or tissue of cancer patients have important implications. For instance, cancer cells require high
ADA activity to metabolize AD for maintaining their division, whereas high AD concentrations can inhibit cell
division [12]. Therefore, an effective treatment strategy is to restrain the deamination function and to elevate the
AD level by inhibiting ADA. 2’-deoxycoformycin (dCF), a potent inhibitor of ADA, has been officially
approved as a drug by the FDA for the treatment of acute lymphoblastic leukemia [13]. However, the proposed
inhibitors exhibit some disadvantages, such as irreversibility, side effects, high inhibition constant (Ki), and
toxicity on different cells [14]. In recent years, natural sources have received growing attention, and most ADA
inhibitors have been isolated from natural plants or structurally modified from natural compounds. Therefore,
identification of ADA inhibitors as potential drug candidates has been a major topic in the field of drug
discovery.
Ultraviolet (UV) spectroscopy is commonly used for screening potential ADA inhibitors [15]. In the UV
method, the ADA reaction is calculated by measuring the decrease in absorbance of the substrate at 265 nm or
increase in absorbance of the product at 248 nm. However, AD and inosine share a very close maximum
absorption wavelength. Thus, exact evaluation of the product formation at the usually selected wavelength is
difficult. In addition, the reaction is uncertain and is frequently interfered by the background color of compounds
4

or plant extracts. The use of the UV method to screen potential ADA inhibitors usually yields false positive or
negatives results. To overcome these drawbacks, many methods, such as radiative method [16], Berthelot method
[17], capillary electrophoresis (CE) [18], high-performance liquid chromatography (HPLC) [19], and aptasensor
method [20], have been developed for screening potential ADA inhibitors. However, these methods still exhibit
some shortcomings. The radioactive method is effective but is inconvenient to use and is environmentally toxic
[16]. The Berthelot method measures the amount of liberated NH3 but suffers from lack of accuracy due to the
interference of exogenous NH3 [17]. Given its high efficiency separations and short analysis times, CE has been
widely used to evaluate ADA activity. However, the measuring sensitivity of the method is very low [18]. HPLC
is also applied in ADA analysis to separate the substrate and the product. Despite its superior repeatability, it
requires intensive sample pretreatment and long running time [19]. The aptasensor approach allows for exclusive
analysis of the substrate AD and indirect detection of ADA activity. Despite its low detection limit, this method
requires many labeled molecules and relevant testing instruments, which result in high costs and limitation for
the general laboratory without these conditions [20]. Therefore, convenient and sensitive methods for the assay
of ADA activity should be developed. UPLC-MS with high sensitivity and selectivity is especially applicable for
detecting enzyme activities [21]. Orbitrap is a new developed mass analyzer in high-resolution mass
spectrometry (HRMS). It promises high mass resolution and mass accuracy, wide dynamic range, and good duty
cycle and sensitivity [22]. Therefore, this work developed a selective, convenient, quick, and sensitive
ultra-high-performance liquid chromatography-Q exactive hybrid quadrupole orbitrap high-resolution accurate
mass spectrometric (UHPLC-Q-Orbitrap HRMS) method for detecting and screening ADA inhibitors. The direct
product (inosine) from the selective substrate (AD) catalyzed by ADA in the incubation was quantitated. The
method not only involves the classic principle of ADA activity assay in vitro but also performs rapid and
sensitive screening of inhibitors and avoids the interferences of the matrix effects as much as possible.
5

2. Experimental
2.1. Chemicals and reagents
ADA, AD, inosine, chlormequat, and erythro-9-(2-hydroxy-3-nonyl)•adenine hydrochloride (EHNA) were
purchased from Sigma–Aldrich (St. Louis, MO, USA). 2’-dCF was purchased from Dalian Meilun Biotech Co.,
Ltd. (Dalian, Liaoning China). The purities of all reference standards were over 98%. HPLC-grade methanol was
obtained from Fisher Co. (Santa Clara, CA, USA). Formic acid was obtained from Tedia Inc. (Fairfield, OH,
USA). Ultra-pure water (18.2 MΩ) was purified using a Milli-Q Academic System (Millipore, Billerica, USA).
All other reagents were of analytical reagent grade.
2.2. Materials
The collected traditional Chinese herbal medicines were purchased from local medicinal markets in Shanghai
in 2017 (Supplementary materials Table S1). The authentication of herbal medicines was confirmed by Professor
Lihong Wu, and the voucher specimens were deposited at the Herbarium of the Shanghai R&D Center for
Standardization of Traditional Chinese Medicine (Shanghai, China).
2.3. Preparation of extracts
The extracts of 26 traditional Chinese herbal medicines were extracted by methanol for inhibitor screening of
ADA. In brief, air-dried Chinese herbal medicine was ground into fine powder in a grinder. Then, 2 g of Chinese
herbal medicine powder was poured into a 100 mL stoppered Erlenmeyer flask, which was then added with 50
mL of methanol. The flask sealed with Parafilm (Parafilm, Chicago, IL, USA) was placed into an ultrasonic
cleaner for ultrasonic extraction of powder for 1 h. The supernatant was acquired by centrifugation at 2300 × g
for 10 min and then evaporated to dryness on vacuum. The dried extract was stored in a closed desiccator for
inhibitor screening.
2.4. Standard stock solutions and quality control samples
6

The primary stock solutions of the product (inosine) and internal standard (IS, chlormequat) were prepared by
dissolving the appropriate amount of each standard substance in 10 mL of methanol to obtain 3.73 and 6.33 mM,
respectively. The stock solutions were prepared separately for calibration curves and quality control (QC)
samples by serially diluting with the initial mobile phase (0.1% formic acid:methanol = 85:15, v/v) to provide
working solutions of desired concentrations. The IS working solution (1.26 μM) was prepared by diluting the
stock solutions with methanol. All solutions were stored at 4 °C and brought to room temperature before use.
2.5. Sample preparation procedure
ADA at 2.3 unit·mL−1 was prepared by dissolving in 20 mM sodium phosphate buffer (pH 7.6) to make stock
solution, and the solution was stored at −80 °C before use. The substrate AD was dissolved in 20 mM sodium
phosphate buffer (pH 7.6) to achieve a concentration of 3.75 mM.
The stock solutions of the Chinese medicine extract were prepared by dissolving an adequate quantity of each
extract (obtained in Section 2.3) in 10% DMSO to obtain 100 mg·mL−1 solutions (calculated by the amount of
crude herb). The stock solutions of the tested positive compounds (EHNA and 2’-dCF) were prepared by
dissolving an adequate quantity of each compound in DMSO to obtain 10 mg·mL−1 solutions. All the stock
solutions were diluted to a series of concentrations with a 20 mM sodium phosphate buffer solution (pH 7.6)
before each experiment. All solutions were filtrated through a 0.22 μm membrane filter before use.
2.6. Chromatography conditions for UHPLC-Q-Orbitrap HRMS
Chromatographic separation was performed on a UHPLC-Q-Orbitrap system (Thermo Fisher Scientific Inc.,
Grand Island, NY, USA). The UHPLC system consisted of a Thermo Scientific Dionex Ultimate 3000 Series RS
pump coupled with a Thermo Scientific Dionex Ultimate 3000 Series TCC-3000RS column compartments and
WPS-3000 autosampler controlled by Chromeleon 7.2 Software. The cooling autosampler was set at 4 °C and
protected from light, and the column heater was set at 40 °C. A Waters ACQUITY UPLC HSS T3 column (2.1 ×
7

100 mm, 1.8 μm) was employed with the temperature set at 40 °C. The mobile phase consisted of A (methanol)
and B (0.1% formic acid) at a flow rate of 0.3 mL·min−1 with isocratic elution of 15% A and 85% B. The
injection volume was 5 μL.
The mass spectrometer Q-Orbitrap system was connected to the UHPLC system via heated electrospray
ionization and controlled by Xcalibur 4.1 software that was used for data capture and analysis. The electrospray
ionization source was operated and optimized in positive ionization mode. The optimized parameters of mass
spectrometry were as follows: capillary temperature, 320 °C; sheath gas (N2) flow rate, 40 arbitrary units;
auxiliary gas (N2) flow rate, 10 arbitrary units; sweep gas flow rate, 0 arbitrary units; spray voltage, 3.50 kV;
S-lens RF level, 50 V; auxiliary gas heater temperature, 300 °C; scan mode, full MS; scan range, 50–500 m/z;
maximum injection time, 200 ms; scan resolution, 70,000 FWHM (m/z/s); and automatic gain control target,
1.0e6. The ions of the product inosine (m/z 269.0880) and the IS (m/z 122.0733) were extracted for quantitative
and qualitative analyses.
2.7. Method validation
The developed UHPLC-Q-Orbitrap HRMS method was validated for selectivity, calibration curve, lowest
limit of quantification (LLOQ), accuracy, precision (inter- and intra-day precisions), matrix effects, and stability
in accordance with the European Medicines Agency’s guideline on bioanalytical method validation and the
literature [23].
Selectivity was confirmed by comparing chromatograms of the blank matrix (0.00736 unit·mL−1 of ADA was
prepared by dissolving in 20 mM of sodium phosphate buffer) pretreated with methanol (containing IS) against
the matrix added to inosine and the IS. The standard curve was obtained by plotting the peak area ratio of the
product to the IS (Y) and applying a weighted (1/y2) least-squares linear regression analysis. The standard
solution of inosine was diluted to seven concentrations with the buffer solution ranging from 0.02 μM to 11.93
8

μM to evaluate the linear relationship of the method. The lowest concentration of inosine threefold the
signal-to-noise ratio was used as the limit of detection (LOD). The LLOQ was defined as the lowest
concentration giving a signal-to-noise ratio at least 10-fold on the calibration curve for which acceptable
accuracy (within 80%–120%) and precision (< 20%) were confirmed.
The precisions of the assay were appraised by performing replicate analysis of the QC samples (n = 5) at five
levels (0.02, 0.05, 0.76, 1.91, and 11.93 μM). Intra-day precision was assessed by repeating the analysis of the
QC samples three times a day, whereas inter-day precision was evaluated by repeating the analysis on three
subsequent days. Relative standard deviation (RSD) was used to determine the precision value calculated from
the observed concentrations (Cobs) in accordance with the following equation: %RSD = [standard deviation
(SD)/Cobs] × 100. Accuracy was expressed by the recovery of the QC samples at five levels (0.02, 0.05, 0.76,
1.91, and 11.93 μM, n=5). The apparent concentrations of these samples were calculated using the calibration
curves. The recovery rates (%) were calculated from the mean value of the observed concentrations (Cobs) and
the theoretical concentrations (Cthe) by using the following equation: % = [Cobs/Cthe] × 100. The matrix effects
were determined by comparing the peak responses of the standard QC samples at three levels (0.02, 0.76, and
11.93 μM, n = 5) dissolved in the blank matrix pretreated with methanol (A) against those dissolved in the initial
mobile phase (B, n = 5). The mean value (A)/(B) × 100 was considered the matrix effect, which is acceptable
when the ratio is more than 85% and less than 115%.
The stability of inosine was conducted on QC samples at three concentration levels (0.02, 0.76, and 11.93 μM)
with five replicates after keeping in the autosampler (4 °C) or refrigerator (4 °C) for 3 days and at ambient
temperature (25 ± 2 °C) for 1 day. The measured concentrations were then compared with those of freshly
prepared QC samples. RSD was used to determine the stability value calculated from the observed
concentrations (Cobs) in accordance with the following equation: %RSD = [standard deviation (SD)/Cobs] × 100.
9

2.8. In vitro anti-adenosine deaminase assay
2.8.1 UHPLC-Q-Orbitrap HRMS
ADA activity assay was carried out to rapidly screen by UHPLC-Q-Orbitrap HRMS using AD as the substrate
under the optimal incubation conditions. The extract of individual traditional Chinese medicines was tested in
concentration ranges between 0.01 and 10 mg·mL−1. Positive compounds (EHNA and 2’-dCF) were tested in
concentration ranges between 0.01 and 10000.0 ng·mL−1. A 60 μL incubation system composed of a 6 μL
solution of the tested traditional Chinese medicine extract and 34 μL of the enzyme solution (final concentration
of 0.00736 unit·mL−1 for ADA) was mixed and pre-incubated for 10 min in a 1.5 mL centrifuge tube. Thereafter,
20 μL of the substrate solution (final concentration of 5.50 μM for AD) was added and incubated for 20 min at
30 °C. The reaction was terminated by immediately adding 180 μL of ice methanol solution (0 °C) containing
3.29 µM IS. The reaction system was diluted by adding 960 μL of ultra-pure water. The tube was tightly capped
and vigorously mixed for 0.5 min using a vortex (Scientific Industries, New York, USA) mixer at maximum
speed. The solution was then centrifuged (15,000 × g, 10 min), and the supernatant was used for the analysis.
The inhibitory activity of the analyte was calculated in accordance with the production of inosine. The formula
of inhibition rate was as follows: Inhibition % = (Ccontrol-Csample)/Ccontrol100. Ccontrol and Csample represent the
production of inosine without and with the test sample, respectively. The assay was carried out in triplicate. The
inhibitory IC50 values of the traditional Chinese medicines on ADA were calculated using the Prism software
(GraphPad Software Inc., San Diego, CA).
2.8.2 Ultraviolet spectroscopy method
The ADA activity assay by the UV method was performed as previously described [24] with slight
modifications. The extract of individual traditional Chinese medicines was tested in concentration ranges
between 0.01 and 10 mg·mL−1. Positive compounds (EHNA and 2’-dCF) were tested in concentration ranges
10

between 0.01 and 10000.0 ng·mL−1. A 150 μL incubation system consisting of 50 μL of the tested traditional
Chinese medicine extract solution and 50 μL of the enzyme solutions (final concentration of 0.38 unit·mL−1 for
ADA) was mixed and then pre-incubated for 10 min in a 96-well UV plate (UV transparent flat bottom, Corning
3635). Then, 50 μL of the substrate solution (final concentration of 0.50 mM for AD) was added. The 96-well
UV plate was immediately placed in a Powerwave XS microplate spectrophotometer reader (Bio Tek
Instruments, Winooski, VT, USA). The absorbance was measured at 265 nm every 10 s for 60 s. The straight line
was drawn with time as the abscissa and light absorption value as the ordinate. The inhibitory activity of the
compound was calculated in accordance with the change in slope of the linear equation. The formula of
inhibition rate was as follows: Inhibition % = (Kcontrol−Ksample)/Kcontrol  100. Kcontrol and Ksample represent the slope
of the linear equation without and with the test sample, respectively. The assay was carried out in six replicates.
The inhibitory IC50 values of the traditional Chinese medicines on ADA were calculated using the Prism
software (GraphPad Software Inc., San Diego, CA).
2.9. Data analysis
All tabulated results were shown as mean ± SD. The IC50 values and the enzyme kinetic parameters (Km and
Vmax) were all calculated from the concentration–response curves via nonlinear regression analysis using the
Prism software.
3. Results and discussion
3.1. UHPLC-Q-Orbitrap HRMS conditions
To achieve suitable retention times, good peak shapes, and high MS sensitivity of inosine and the IS, various
mobile phase conditions were studied. Methanol or acetonitrile as organic phase and water as polar phase with
different concentrations of ammonium formate, ammonium acetate (from 1 mM to 10 mM), and acetic and
formic acid (from 0.05 to 0.5%) were examined. Finally, methanol–water containing 0.1% formic acid was
11

employed as the mobile phase to obtain good peak shapes for inosine and the IS. Moreover, an optimal isocratic
elution and 0.3 mL·min−1 flow rate provided the improved peak shape of inosine and the IS with the column
temperature set at 40 °C. At the aforementioned UHPLC conditions, the baseline separation of inosine and the IS
was achieved on an HSS T3 column, and both were eluted at 1.53 and 0.95 min, respectively (Fig. 2B). The total
analysis time for a single run was completed within 2 min.
The molecular weight of inosine (C10H12N4O5, M.W. 268.23) was approximately 1.0 Da greater than that of
AD (C10H13N5O4, M.W. 267.24). When inosine and AD did not reach baseline separation, the interference peak
of AD isotope was found in the extracted ion chromatogram of inosine by the low-resolution model
(Supplementary materials Fig. S1A). The challenge, however, could be overcome by high-resolution MS
perfectly. It can provide ion spectra with accurate mass measurement. The interference peak of AD isotope was
undetected in the extracted ion chromatogram of inosine (Supplementary materials Fig. S1B). Therefore, a
proper separation was achieved for inosine and the IS within 2 min via simple isocratic elution, which ignored
the interference peak of AD isotope by UHPLC-Q-Orbitrap HRMS. However, the complex gradient elution
conditions needed at least 6 min to achieve the proper separation of AD, inosine, and IS by UHPLC coupled
with low-resolution MS (data not shown).
3.2. Method validation
The chromatographic separation is shown in Fig. 2. No significant interfering peaks were observed at the
retention times of either analytes or internal standard in the blank matrix. The calibration curve showed a good
linear behavior over the inosine concentration range of 0.02–11.93 μM (y = 0.312x + 0.00603, R2= 0.9951,
weighed: 1/y2). The LOD (S/N = 3) of inosine was 0.013 μM, and the LLOQ (S/N = 10) was 0.02 μM for the
product. The LLOQ is appropriate for the quantitative detection of inosine in the enzymatic studies.
The results of intra- and inter-day precisions, accuracy, matrix effect, and stabilities of the method are
12

summarized in Table 1. The RSD values of intra- and inter-day precisions ranged from 2.14% to 7.17% and from
2.55% to 8.99%, respectively. The average recoveries of inosine at five concentrations ranged from 85.69% ±
1.13% to 111.3% ± 0.85%. These results suggested that the method exhibited satisfactory precision and accuracy.
The matrix effects for inosine ranged from 101.64% ± 3.24% to 107.12% ± 7.05%. The data suggested that no
ionization enhancement or suppression was observed for inosine under the present conditions. Inosine was stable
after being placed at room temperature for 24 h and kept in the autosampler or refrigerator (4 °C) for 3 days.
3.3. Influence of temperature on the enzymatic reaction
The effect of temperature on the enzymatic reaction was studied by varying the temperature in the range of
20 °C–45 °C (Fig. 3A). No obvious influence of temperature from 30 °C to 45 °C was observed on ADA
reaction. Therefore, the temperature for enzymatic reaction was selected at 30 °C.
3.4. Optimization of reaction time, substrate concentration, and ADA concentration
The enzyme concentration, substrate concentration, and reaction time of the assay should be appropriate prior
to its development. To ensure the utilization of the initial rate velocities for the calculation of enzyme constants
in accordance with steady-state kinetics, the optimum reaction time, substrate concentration, and ADA
concentration should be within the linear region of the formation of the product. Three reaction functions were
generated by monitoring the product as functions of reaction time, substrate concentration, and enzyme
concentration. The reaction time was set as 20 min for the ADA assay (Fig. 3B). The formation of inosine
showed good linear equation of y = 0.186x + 0.779 (R2 = 0.987) for ADA assay (Fig. 3C) when the substrate
concentration of AD ranged from 1.87 μM to 37.42 μM. Thus, the substrate concentration of AD was selected at
3.74 μM. The formation of inosine showed a good linear equation of y = 1325.272x +1.321 (R2 = 0.946) for
ADA assay (Fig. 3D) when the enzyme concentration of ADA ranged from 0.0005 unit·mL−1 to 0.018 unit·mL−1.
Accordingly, the enzyme concentration was selected at 0.007 unit·mL−1. The product inosine could be quantified
13

sensitively, and even 99% of ADA enzyme activity was inhibited.
3.5. Michaelis constant (Km) of ADA
From Supplementary materials Fig. S2, the linear Eadie–Hofstee plots (II) were found to exhibit
Michaelis–Menten kinetics for ADA-catalyzed enzyme reaction. The values of Km and Vmax for ADA were 81.70
± 13.82 µM and 779.2 ± 43.99 µM·min−1·unit−1, respectively. The values of Km ranged from 25 µM to 550 µM
in previous reports because Km varies with the reaction conditions, analytical methods, and enzyme sources used
[25-28]. The value of Km in this assay is highly consistent with that obtained from the UV method (89 µM),
which was used under similar reaction conditions (such as substrate, reaction temperature, and pH of buffer) [28].
This result indicated no significant change in the enzymatic property of the optimized incubation system.
3.6. Detection of inhibitory activity of traditional Chinese medicines against enzymes
The ADA inhibitory activities of traditional Chinese medicines and model compounds were evaluated in vitro
by using the UHPLC-Q-Orbitrap HRMS and UV methods. As shown in Table 2, EHNA and 2’-dCF exhibited
exceedingly strong inhibition on ADA, which was in good agreement with previous reports [29, 30]. Among the
tested traditional Chinese medicines, 13 exhibited different potential inhibition activities on ADA. Ligustrum
lucidum and Rheum palmatum showed strong inhibitory effects with IC50 values below 1 mg·mL−1 on ADA, and
Salvia chinensis showed exceedingly strong inhibitory effects with IC50 values below 0.1 mg·mL−1 on ADA.
To interpret the correlation of the two methods, a bivariate correlation analysis for the IC50 values obtained
from the UHPLC-Q-Orbitrap HRMS and UV methods was conducted (Supplementary materials Fig. S3). The
IC50 values of the model compounds (EHNA and 2’-dCF) and 13 traditional Chinese medicines obtained from
the two methods (Table 2) were used in the correlation analysis. A significant linear correlation was found
between the UHPLC-Q-Orbitrap HRMS and UV methods with correlation factors (r) of 0.9654 (p< 0.001) and
the linear equation of y = 1.163x + 0.0963 (R2 = 0.9321). This result indicated the reliability of the newly
14

established UHPLC-Q-Orbitrap HRMS method. Enzymatic reaction was a dynamic process, and the UV method
did not end the reaction immediately. Thus, the standard deviations (SD) determined by the UV method were
generally higher than that by the UHPLC-Q-Orbitrap HRMS method (Table 2). Therefore, the repeatability of
the UV method was lower than that of the UHPLC-Q-Orbitrap HRMS method. In addition, the enzyme and
substrate concentrations used in the UV method (ADA: 0.38 unit·mL−1, AD: 0.50 mM) were several folds higher
than those in the UHPLC-Q-Orbitrap HRMS method (ADA: 0.00736 unit·mL−1, AD: 5.50 μM). In
UHPLC-Q-Orbitrap HRMS, the potential instability of the dynamic enzymatic reaction was avoided (the
enzymatic reaction was terminated by methanol) along with the interference of the background color of
compounds or plant extracts. Therefore, the newly developed UHPLC-Q-Orbitrap HRMS method is sensitive
and reliable and can be used to avoid false negative results.
4. Conclusion
A UHPLC-Q-Orbitrap HRMS method was developed using AD as the substrate for determining the activity of
ADA inhibitors. The method achieved a proper separation for the product inosine and the IS within 2 min by
isocratic elution on an efficient HSS T3 column without any matrix effect. The method is fast, sensitive, accurate,
and repeatable. It also exhibits smaller total reaction volume, shorter running time, higher sensitivity, and lower
cost than the conventional methods. Thus, this method is suitable for the high-throughput screening of potential
ADA inhibitors from natural medicinal plants.
Acknowledgments
This work was supported by the Key projects of National Natural Science Foundation of China (Grant No.
81530101), the National Nature Science Foundation of China (Grant No. 81703681), the Shanghai Sailing
Program (Grant No. 17YF1419800), and the Budget Project of Shanghai University of Traditional Chinese
Medicine (Grant No. 2016YSN12) awarded to Professors Chang-hong Wang and Ping Liu for financial support
15

of this study.
Conflict of interest
The authors declare no conflict of interest.
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Figure legends
Fig. 1. The irreversible hydrolytic deamination of adenosine (AD) and deoxyadenosine to produce inosine and
2’-deoxyinosine by adenosine deaminase.
Fig. 2. Extracted ion chromatograms of the product inosine and chlormequat (internal standard, IS), A: a blank
matrix spiked with IS; B: a blank matrix spiked with the analyte and IS.
Fig. 3. Influence of temperature (A), incubation time (B), substrate concentration (C) and ADA concentration (D)
on the production of inosine converted from adenosine.
20

21

Table 1. Results of method validation for the UHPLC-Q-Orbitrap HRMS analysis of inosine.
Measured concentration
Accuracy
(%)
Precision (RSD%)
Matrix effect (%)
Stability (RSD%)
Nominal level (μM)
(μM)
Intra-day
7.17
Inter-day
(RT)
(4℃)
7.64
/
0.02 (LLOQ)
0.05 (QCL)
0.018±0.0004
0.048±0.0034
0.85±0.0065
2.09±0.04
89.17±2.04
97.27±6.86
111.30±0.85
109.33±1.99
85.69±1.13
8.99
6.54
3.29
5.36
2.55
107.12±7.05
8.36
/
5.23
/
0.76 (QCM)
2.23
101.64±3.24
/
2.31
/
3.02
/
1.91 (QCH)
3.61
11.93 (ULOQ)
RT: Room Temperature
10.23±0.13
2.14
103.00±2.68
2.27
3.13
22

Table 2. Inhibitory activities (IC50) of the tested traditional Chinese medicines and positive compounds
investigated against ADA by UHPLC-Q-Orbitrap HRMS and UV methods (mean ±SD).
IC50 for ADA (mg·mL-1)
Natural extract/Compounds
UHPLC-Q-Orbitrap HRMS (n=3)
2.27±0.04
1.76±0.03
1.85±0.01
4.38±0.07
2.96±0.02
1.65±0.03
2.32±0.03
1.41±0.004
0.72±0.007
0.05±0.003
2.02±0.02
0.57±0.006
2.67±0.01
＞10
UV (n=6)
2.56±0.04
2.65±0.13
1.20±0.14
5.33±0.07
3.55±0.11
2.48±0.12
3.02±0.12
1.50±0.04
1.37±0.04
0.13±0.003
2.66±0.09
0.78±0.01
2.86±0.05
＞10
Bupleurum chinense DC.
Ligusticum chuanxiong Hort.
Angelica sinensis (Oliv.) Diels.
Sophora flavescens Ait.
Artemisia capillaris Thun.
Artemisia argyi LevL. et Vant.
Atractylodes macrocephala Koidz.
Isatis indigotica Fort.
Ligustrum lucidum Ait .
Schisandra chinensis (Turcz.) Baill.
Gardenia jasminoides Ellis
Rheum palmatum L.
Cordyceps sinensis (Berk.) Sacc.
Sophora tonkinensis Gagnep.
Polyporus umbellatus (Pers.)Frie.
Isatis indigotica Fort.
＞10
＞10
＞10
＞10
Prunus persica(L.) Batsch
Gynostemma pentaphyllum (Thunb.) Makino.
Paeonia lactiflora Pall.
＞10
＞10
＞10
＞10
＞10
＞10
Salvia miltiorrhiza Bge.
＞10
＞10
Stephania tetrandra S. Moore
Coptis chinensis Franch.
＞10
＞10
＞10
＞10
Astragalus membranaceus (Fisch.) Bge.
Curcuma Longa L
＞10
＞10
＞10
＞10
Eupolyphaga sinensis Walker
Glycyrrhiza uralensis Fisch.
EHNA
＞10
＞10
＞10
＞10
6.47E-05±5.77E-07
1.14E-07±7.28E-09
7.12E-05±3.07E-06
2.05E-07±1.41E-08
2’-dCF
23