1 s2.0 S0021967313008546 Main
1 s2.0 S0021967313008546 Main
1 s2.0 S0021967313008546 Main
Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma
a r t i c l e i n f o a b s t r a c t
Article history: In this study, a rapid and sensitive analytical method was developed for the determination of 20
Received 9 April 2013 nucleobases, nucleosides and nucleotides in Ziziphus plants at trace levels by using hydrophilic inter-
Received in revised form 20 May 2013 action ultra-high performance liquid chromatography coupled with triple-quadrupole tandem mass
Accepted 29 May 2013
spectrometry (HILICUHPLC-TQ-MS/MS) in multiple-reaction monitoring (MRM) mode. Under the
Available online 6 June 2013
optimized chromatographic conditions, good separation for 20 target compounds were obtained on
a UHPLC Amide column with sub-2 m particles within 10 min. The overall LODs and LOQs were
Keywords:
between 0.113.12 ng mL1 and 0.2912.48 ng mL1 for the 20 analytes, respectively. It is the rst report
Nucleobases
Nucleosides
about simultaneous analysis of nucleobases, nucleosides and nucleotides in medicinal plants using
Nucleotides HILICUHPLC-TQ-MS/MS method, which affords good linearity, precision, repeatability and accuracy.
HILICUHPLC-TQ-MS/MS The developed method was successfully applied to Ziziphus plant (Z. jujuba, Z. jujuba var. spinosa and Z.
Ziziphus mauritiana) samples. The analysis showed that the fruits and leaves of Ziziphus plants are rich in nucle-
osides and nucleobases as well as nucleotides, and could be selected as the healthy food resources. Our
results in present study suggest that HILICUHPLC-TQ-MS/MS method could be employed as a useful tool
for quality assessment of the samples from the Ziziphus plants as well as other medicinal plants or food
samples using nucleotides, nucleosides and nucleobases as markers.
2013 Elsevier B.V. All rights reserved.
0021-9673/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.chroma.2013.05.074
148 S. Guo et al. / J. Chromatogr. A 1301 (2013) 147155
induced by the phosphate moieties rendering their retention a The fruits and leaves of Ziziphus jujuba (ZJ), Z. jujuba var. spinosa
challenge [30]. Thus, to avoid the above problem, the ion-pairing (ZS) and Z. mauritiana (ZM) were collected from different habitats
reversed-phase liquid chromatography (IP-RP-LC) method was in China, and the detailed information is listed in Table 1. Their
developed with the C18 columns and ion-paring agents, and the botanical origins were identied by the corresponding author, and
successful retention of these polar compounds was obtained in voucher specimens were deposited at the Herbarium in Nanjing
this way [3032]. However, most of the ion-pairing agents could University of Chinese Medicine, China. After collection, the entire
decrease the sensitivity of the MS detector [31]. Alternatively, the fruits and leaves were dried at 45 C, respectively, which is better
highly polar compounds can obtain good retention and separation consistent with the current production facts of the Ziziphus plants.
on hydrophilic interaction chromatography (HILIC). In contrast
to RP-HPLC, HILIC separation is based on the strong hydrophilic 2.2. Preparation of standard solutions
interaction of polar compounds with the hydrophilic polar sta-
tionary phase [33]. It is suitable for the separation of a broad Standard stock solutions (100 g mL1 ) were prepared by dis-
spectrum of highly polar compounds, including peptides, amino solving about 10 mg of individual compounds in 100 mL of water.
acids, carbohydrates as well as many other biologically important These solutions were diluted with water to a series of appropriate
compounds [3438]. Recently, HILIC method was also successfully concentrations and used to construct calibration curves. All these
applied to analyze nucleobases, nucleosides as well as nucleotides solutions were stored in the refrigerator at 4 C before analysis and
in herbal medicines and biological uids [10,3942]. Although were ltered through a 0.22 m membrane prior to injection.
the above HILIC methods were powerful, they still showed some
limitations such as limited analytes detected, extended analysis 2.3. Preparation of sample solutions
time, and poor selectivity.
Thanks to the progress of chromatographic technique, ultra- The dried fruits of ZJ, ZS and ZM were divided into sarcocarp,
high performance (or ultra-performance) liquid chromatography hardcore and seed parts, which were further pulverized to homoge-
(UHPLC or UPLC) was developed by using columns containing par- neous powder (40 mesh), respectively. Likewise, the leaf powders
ticles with a diameter of sub-2 m, which shortened the analysis of ZJ and ZS were obtained according to the same method. The dried
time and increased the peak resolution, capacity and sensitivity, powder (1.0 g) was weighed accurately into a 50 mL conical ask
especially when it is coupled with tandem mass spectrometry with stopper, and 40 mL water was added accurately. After weigh-
(MS/MS) [43]. Recently, the method combined UHPLC with tandem ing the lled ask accurately, ultrasonication (40 kHz) was carried
mass spectrometry have been applied to determine nucleotides, out at room temperature for 30 min, then weighing again, and the
nucleosides and nucleobases, while most of them were focused on same solvent (water) was added to compensate for the lost weight
the samples from biological uids. during the extraction as needed. After centrifugation (15,000 x g,
Here we report the development, validation, and application 10 min), the supernatant was stored at 4 C and ltered through
of a fast and sensitive HILICUHPLC method for separation of a 0.22 m polytetrauoroethylene lter before injection into the
nucleotides, nucleosides and nucleobases in samples from Zizi- UHPLCMS/MS system for analysis.
phus plants. In addition, due to the high sensitivity and wide
linear dynamic range for quantitation, a triple quadrupole mass 2.4. HILICUHPLC-TQ-MS analysis
spectrometer (TQ-MS) was used for the qualitative and quantita-
tive determination of these compounds in this assay. The present UHPLC was performed by using a Waters ACQUITY UPLC system
method is more applicable to the determination of nucleotides, (Waters, Milford, MA, USA), equipped with a binary solvent delivery
nucleosides and nucleobases originated from plant samples which system and an auto-sampler. Hydrophilic interaction chromato-
always showing different complexity from bio-samples such as graphic separation was performed on an ACQUITY UPLC BEH Amide
blood or urine. column (2.1 mm x 100 mm, 1.7 m) equipped with an ACQUITY
UPLC BEH Amide 1.7 m VanGuard Pre-column. In addition, a BEH
HILIC column (2.1 mm x 100 mm, 1.7 m) was also used for the
2. Experimental optimization of the separation method. The mobile phase was com-
posed of A (0.8% acetic acid and 10 mM ammonium acetate in
2.1. Chemicals and materials aqueous solution) and B (0.1% acetic acid in acetonitrile) with a
gradient elution: 06 min, 10% A; 69 min, 10%40% A; 912 min:
Acetonitrile (HPLC-grade) was purchased from Merck (Darm- 40%50% A, and the column was equilibrated for 6 min in the initial
stadt, Germany), and deionized water (H2 O) was puried by a conditions. The ow rate of the mobile phase was 0.4 mL min1 , and
Milli-Q water purication system (Millipore, Billerica, MA, USA). the column temperature was maintained at 35 C with a column
Other reagent solutions, such as ammonium acetate and acetic temperature oven. Two cycles of strong (20% acetonitrile, 400 L)
acid, were of analytical grade (Sino Pharm Chemical Reagent Co., and weak (90% acetonitrile, 600 L) solvents were used to wash
Ltd., Shanghai, China). Chemical standards of thymine, thymidine, the injecting system between injections, and these washing sol-
2 -deoxyuridine, adenosine, 2 -deoxyinosine, xanthine, inosine, vents did not transfer to the column. The injection volume was
2 -deoxyguanosine, cytidine, guanosine, guanosine 3 ,5 -cyclic 1 L. The column eluent was directed to the mass spectrometer.
monophosphate (cGMP), 2 -deoxyadenosine-5 -monophosphate All the analyses were operated by MassLynxTM XS Software.
and cytidine-5 -monophosphate (CMP) were from Sigma Chemical Mass spectrometry was performed on a Waters Xevo TQ tan-
Co. (St. Louis, MO, USA). Chemical standards of adenine, hypoxan- dem quadrupole mass spectrometer (Micromass MS Technologies,
thine, uridine, guanine and adenosine 3 ,5 -cyclic monophosphate Manchester, UK) using an ESI source operated in positive ion mode.
(cAMP) were purchased from the National Institute for the Control The parameters in the source were set as follows: capillary volt-
of Pharmaceutical and Biological Products (Beijing, China). Refer- age 3.0 kV; source temperature 150 C; desolvation temperature
ence compounds of 2 -deoxyadenosine and 2 -deoxycytidine were 550 C; cone gas ow 50 L h1 ; desolvation gas ow 1000 L h1 . Data
obtained from Aladdin Chemical Co. (Nanjing, China). The purity were collected in multiple reaction monitoring (MRM) or select
of each compound was above 98%, determined by HPLC analysis. ion monitoring (SIM) mode by screening parent and daughter ions
The chemical structures of these reference compounds are shown simultaneously. The cone voltage and collision energy were opti-
in Fig. 1. mized individually for each target compound and selected values
S. Guo et al. / J. Chromatogr. A 1301 (2013) 147155 149
Thymine (1) Thymidine (2) 2-deoxyuridine (3) 2-deoxyadenosine (4) Adenine (5) Hypoxanthine (6)
Uridine (7) Adenosine (8) 2-deoxyinosine (9) Xanthine (10) 2-deoxycytidine (11) Inosine (12)
Guanine (13) 2-deoxyguanosine (14) Cytidine (15) Guanosine (16) cAMP (17) cGMP (18)
Fig. 1. Chemical structures of investigated nucleobases, nucleosides and nucleotides. In these analytes, 1, 5, 6, 10, and 13 are nucleobases; 24, 79, 11, 12, and 1416 are
nucleosides; 1720 are nucleotides.
are given in Table 2. Dwell time was automatically set by the soft- 2.5.2. Precision, repeatability and stability
ware. Intra- and inter-day variations were chosen to determine the
precision of the developed method. In intra-day variability test, the
mixed standards solutions were analyzed for six replicates within a
2.5. Validation of the method day; while in inter-day variability test, the solutions were examined
in duplicates for consecutive three days. To conrm the repeatabil-
The method was validated for linearity, limits of detection and ity, the same weight of ZJF1, ZJS1, ZJH1 and ZJL1 were mixed to
quantication (LODs and LOQs), precision (inter-day and intra-day generate the quality control sample (QC sample), and by using the
precision), reproducibility, and stability following the Interna- QC sample, the six sample solutions were independently prepared
tional Conference on Harmonization (ICH) guideline [44] and some and analyzed. One of the QC sample solutions was stored at 20 C
reports on determination analysis [31,45,46]. and analyzed at 0, 2, 4, 8, 12, and 24 h, respectively to evaluate their
stability. All these variations were expressed by relative standard
2.5.1. Calibration curves, LOD and LOQ deviation (RSD).
Calibration curves were constructed from peak areas of the
reference standards versus their concentrations. Each calibration 2.5.3. Recovery
curve was performed with at least six appropriate concentra- A recovery test was applied to evaluate the accuracy of this
tions in duplicate. Linearity evaluation of calibration curve was method. It was performed by adding the corresponding marker
accomplished by applying the lack-of-t test using SPSS 16.0 soft- compounds with three different levels (high, middle and low) to
ware (SPSS, Chicago, IL). The LOD and LOQ for each analyte were 0.5 g of QC sample which had previously been analyzed in the
determined at the signal-to-noise ratio (S/N) of about 3 and 10, repeatability test and the average content determined in repeat-
respectively. ability assay were selected as the original amount of QC sample
150 S. Guo et al. / J. Chromatogr. A 1301 (2013) 147155
Table 1
The information of the tested samples.
for the recovery test. The spiked samples were then extracted, nucleotides, the slope comparison method was used to evaluate
processed, and quantied in accordance with the methods men- the matrix effect for this study [47,48]. The sample extracts, which
tioned above, and triplicate experiments were performed on were spiked with appropriate amounts of standards as done for the
each level. The average recoveries were estimated by the for- apparent recovery measurement, were used to construct standard
mula: recovery (%) = [(amount found original amount)/amount addition calibration curves. Afterwards, the slopes of the calibration
added] x 100%. curves from the standard addition experiments were compared
with the slopes obtained from the pure aqueous standards on the
2.5.4. Matrix effect same concentration levels. The slope ratios (slope matrix/slope sol-
The matrix effect was dened as the ion suppression or enhance- vent) of 1 indicates that matrix does not signicantly suppress or
ment of the ionization of analytes. Because it is very difcult to enhance the response of MS, otherwise denoting ionization sup-
nd blank matrix samples free of nucleobases, nucleosides and pression (<1) or enhancement (>1) [47].
Table 2
MS parameters of 20 investigated nucleotides, nucleosides and nucleobases.
Analyte [M+H]+ (m/z) MRM transitions/SIM Cone voltage (V) Collision energy (eV) Retention time (min)
2.6. Data processing and statistical analysis tandem mass spectrometer [49,50], while few for simultaneous
separation of nucleotides, nucleobases and nucleosides in a single
Data were processed using the TargetLynx application manager run, like presented in this study.
for the quantication of compounds. Principle component analysis
(PCA) was performed using SPSS 16.0 software. 3.2. Optimization of TQ-MS conditions
within the test ranges. Since the eluent of the proposed HILIC is the developed HILICUHPLC-TQ-MS method was sensitive, repeat-
mainly the aqueous organic solution which enhances the analyte able, and accurate for the quantication analysis of these target
ionization with ESI process, the method showed high sensitivity compounds.
with the LODs and LOQs from 0.11 to 3.12 ng mL1 and 0.29 to
12.48 ng mL1 which are much lower than those obtained with 3.4. Application to the analysis of real samples
the IP-RP-LC method [31] and also lower than those obtained
with the method using the same column while detected by Q- In order to show the utility of the method in proling studies
Tof MS detector [49]. The overall intra- and inter-day variations of nucleotides, nucleosides and nucleobases, an application in real
(RSDs) based on the peak areas of the analyte were in the range of samples was performed. Total 34 batches of samples from three
0.63%4.84% and 1.56%6.37%, respectively. The repeatability and Ziziphus plants (ZJ, ZS and ZM) with different parts including sar-
stability present as RSD were from 0.93% to 7.14% and 2.44% to cocarp, seed, hardcore and leaf were analyzed with the established
6.28%. The overall recoveries were between 90.1% and 103.1% with HILICUHPLC-TQ-MS method. For the seed sample of ZJ, only the
RSD between 2.23% and 6.32%. The slope ratio values of matrix sample from Wuhu, Anhui province was analyzed because no seeds
curve to neat solution curve were between 0.90 and 1.06, indi- were found in other samples. Table S2 shows the concentrations of
cating that the matrix effect on the ionization of analytes was the individual as well as the total contents of these compounds ana-
not obvious under these conditions. These results revealed that lyzed. It was found that these Ziziphus samples except those from
A
10 0 thymine: 127.0 > 127.0 10 0 2-deoxycytidine: 228.0 > 111.9
% 1 1.02e5
%
11 1.83e5
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
2-deoxyadenosine-5-monophosphate:
10 0 2-deoxyinosine: 253.0> 136.9 10 0 332.0> 135.9
9 1.45e5 19 1.70e4
% %
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
10 0 10 0
10 xanthine: 153.0> 153.0
1.05e4
20 CMP: 324.0> 111.9
1.60e4
% %
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
Fig. 2. HILICUHPLC-TQ-MS/MS MRM and SIM chromatograms of mixture standards (A) and the QC sample (B). The compounds numbers on the chromatograms were thymine
(1), thymidine (2), 2 -deoxyuridine (3), 2 -deoxyadenosine (4), adenine (5), hypoxanthine (6), uridine (7), adenosine (8), 2 -deoxyinosine (9), xanthine (10), 2 -deoxycytidine
(11), inosine (12), guanine (13), 2 -deoxyguanosine (14), cytidine (15), guanosine (16), adenosine 3 ,5 -cyclic monophosphate (17), guanosine 3 ,5 -cyclic monophosphate
(18), cytidine-5 -monophosphate (19) and 2 -deoxycytidine-5 -monophosphate (20), respectively.
S. Guo et al. / J. Chromatogr. A 1301 (2013) 147155 153
B
10 0 10 0
1 thymine: 127.0> 127.0 11 2-deoxycytidine: 228.0 > 111.9
1.53e5
% 4.46e4 %
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
10 0 10 0
4 2-deoxyadenosine: 252.0> 135.9
6.87e4
14 2-deoxyguanosine: 268.1> 152.0
1.64e5
% %
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
10 0
10 0
5 adenine: 136.0> 136.0
4.12e5
15 cytidine: 244.0> 112.0
2.29e6
% %
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
10 0
10 0
6 hypoxanthine: 137.0> 137.0 16 guanosine: 284.1> 152.0
1.15e6
% 1.59e6 %
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
10 0 10 0
8 adenosine: 268.1> 136.0
1.13e6
18 cGMP: 346.0> 152.0
6.60e4
% %
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
2-deoxyadenosine-5-monophosphate:
10 0 9 2-deoxyinosine: 253.0> 136.9 10 0
19 332.0> 135.9
3.21e4
% % 1.14e4
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
CMP: 324.0> 111.9
10 0
10 0
10 xanthine: 153.0> 153.0 20 1.32e4
% 1.52e4 %
0 T im e 0 T im e
-0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00 -0.00 2.0 0 4.0 0 6.0 0 8.0 0 10 .00 12 .00
Fig. 2. (Continued).
Table 3
Regression equation, correlation coefcients, linearity ranges and limit of detection (LOD) and quantitation (LOQ) of the target compounds.
Analyte Calibration curvesa r2 Linear range (g mL1 ) LOD (ng mL1 ) LOQ (ng mL1 )
Table 4
Precision, repeatability, stability, recovery and matrix effect of the 20 target compounds.
Analyte Precision (RSD, %) Repeatability (RSD, Stability (RSD, %, Recovery (%, n = 3) Matrix effecta
%, n = 6) n = 6)
hardcore parts (ZJH1 and ZSH14) were rich in nucleobases, nucle- respectively, while they could not be detected in the samples from
osides and nucleotides, especially those from leaf parts (ZJL14 and other parts.
ZSL14) which have not been researched or utilized until now. The
above ndings suggested the leaves of these Ziziphus plants could 3.5. PCA of the samples
be selected as the promising natural sources for future industrial
research of nucleosides, nucleobases and nucleotides. As for the To evaluate the variation between the different parts of these
different species, the contents of these analytes in sarcocarp of ZS Ziziphus plants, PCA was performed on the basis of the contents
were lower than those of ZJ and ZM. As for the individual com- of 20 tested compounds from UHPLC proles. The rst three prin-
pound determined in the experiments, the remarkable differences cipal components (PC 1, PC 2 and PC 3) with >62% of the whole
were also observed. For example, the contents of ribonucleosides variance, were extracted for analysis. The remaining principal com-
in different parts of Ziziphus plants were obviously larger than ponents, which had a minor effect on the model, were discarded.
those of deoxynucleosides, and the latter was found mainly in the The components loading matrix is shown in Table S3. According to
leaves. As for the nucleotides, it was found that CMP mainly exist in their loadings, most of the analytes showed good correlation with
the seeds, while 2 -deoxyadenosine-5 -monophosphate was only the rst three principal components. The sample scatter plots are
detected in trace amount in the samples of leaf and some fruits. Fur- shown in Fig. 3, where each sample is represented as a marker. It
thermore, it was determined that the cyclic nucleotides including was noticeable that the samples were clustered into two domains
cAMP and cGMP are the predominant constituents in the sarcocarp (seed vs sarcocarp, leaf vs sarcocarp, seed vs leaf), respectively.
samples and the highest content were of 984 g g1 and 675 g g1 , These results indicated that the samples from different parts of
Fig. 3. The scatter plots obtained by PCA of the samples. Sarcocarp ( ), seed () and leaf ( ) from Z. jujuba; sarcocarp ( ), seed () and leaf ( ) from Z. jujuba var.
spinosa; sarcocarp ( ), seed () and leaf ( ) from Z. mauritiana.
S. Guo et al. / J. Chromatogr. A 1301 (2013) 147155 155
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