Journal of Chromatography A, 1301 (2013) 147155

Contents lists available at SciVerse ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Hydrophilic interaction ultra-high performance liquid

chromatography coupled with triple quadrupole mass spectrometry
for determination of nucleotides, nucleosides and nucleobases in
Ziziphus plants
Sheng Guo a,b , Jin-ao Duan a, , Dawei Qian a , Hanqing Wang a,c , Yuping Tang a , Yefei Qian a ,
Dawei Wu a , Shulan Su a , Erxin Shang a
a
Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, China
b
Research Center of Basic Medical College, Nanjing University of Chinese Medicine, Nanjing 210023, China
c
College of Pharmacy, Ningxia Medical University, 1160 Shengli Street, Yinchuan, Ningxia 750004, China

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.

1. Introduction in nucleosides and nucleobases [15]. However, besides its fruit,

Nucleosides, nucleotides and nucleobases, a group of highly
active compounds, are involved in the regulation and modulation
of various physiological processes in human body [1,2], and exhibit
multiple bioactivities, such as anti-platelet aggregation [3], anti-
arrhythmic [4], anti-oxidant [5], anti-seizure [6], and anti-tumor
effects [7]. Accordingly, these compounds have held attraction in
the scientic eld recently and many of them have been found in
the medicinal herbs and foods, such as Cordyceps [8], Ganoderma [9],
geosaurus and leech [10], Panax notoginseng [11], Fritillaria cirrhosa
[12], Mactra veneriformis [13], mushrooms [14].
Our previous study showed that the ripe fruit of Ziziphus jujuba,
a medicine and food dual purposes plant used in China, is rich
Corresponding author. Tel.: +86 25 8581 1116; fax: +86 25 8581 1116.
E-mail address: dja@njutcm.edu.cn (J.-a. Duan).
these ingredients in other parts of this plant such as seed, hardcore
and leaf have never been investigated, not to mention the other
medicinal plants of Ziziphus including Z. jujuba var. spinosa and Z.
mauritiana. Therefore, the purpose of this study was to establish an
approach for simultaneous detection and determination of these
three classes of compounds (nucleosides, nucleotides and nucle-
obases) in Ziziphus plants.
To date, several analytical methods including reverse
phase-high performance liquid chromatography (RP-HPLC)
[9,11,1622], ultra-high performance liquid chromatography
(UHPLC) [15,23,24], and capillary electrophoresis (CE) or capillary
electrochromatography (CEC) [2529] have been developed for the
quantication of nucleosides and their analogs in herbal medicines
and biological uids using either ultraviolet detector (UV) or mass
spectrometric detection (MS). It is worth noting the obstacles
of RP-HPLC method as the classical and most frequently used
approach to the analysis of nucleotides due to the high polarity

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
challenge [30]. Thus, to avoid the above problem, the ion-pairing
reversed-phase liquid chromatography (IP-RP-LC) method was
developed with the C18 columns and ion-paring agents, and the
successful retention of these polar compounds was obtained in
this way [3032]. However, most of the ion-pairing agents could
decrease the sensitivity of the MS detector [31]. Alternatively, the
highly polar compounds can obtain good retention and separation
on hydrophilic interaction chromatography (HILIC). In contrast
to RP-HPLC, HILIC separation is based on the strong hydrophilic
interaction of polar compounds with the hydrophilic polar sta-
tionary phase [33]. It is suitable for the separation of a broad
spectrum of highly polar compounds, including peptides, amino
acids, carbohydrates as well as many other biologically important
compounds [3438]. Recently, HILIC method was also successfully
applied to analyze nucleobases, nucleosides as well as nucleotides
in herbal medicines and biological uids [10,3942]. Although
the above HILIC methods were powerful, they still showed some
limitations such as limited analytes detected, extended analysis
time, and poor selectivity.
Thanks to the progress of chromatographic technique, ultra-
high performance (or ultra-performance) liquid chromatography
(UHPLC or UPLC) was developed by using columns containing par-
ticles with a diameter of sub-2 m, which shortened the analysis
time and increased the peak resolution, capacity and sensitivity,
especially when it is coupled with tandem mass spectrometry
(MS/MS) [43]. Recently, the method combined UHPLC with tandem
mass spectrometry have been applied to determine nucleotides,
nucleosides and nucleobases, while most of them were focused on
the samples from biological uids.
Here we report the development, validation, and application
of a fast and sensitive HILICUHPLC method for separation of
nucleotides, nucleosides and nucleobases in samples from Zizi-
phus plants. In addition, due to the high sensitivity and wide
linear dynamic range for quantitation, a triple quadrupole mass
spectrometer (TQ-MS) was used for the qualitative and quantita-
tive determination of these compounds in this assay. The present
method is more applicable to the determination of nucleotides,
nucleosides and nucleobases originated from plant samples which
always showing different complexity from bio-samples such as
blood or urine.
2. Experimental
2.1. Chemicals and materials
Acetonitrile (HPLC-grade) was purchased from Merck (Darm-
stadt, Germany), and deionized water (H2 O) was puried by a
Milli-Q water purication system (Millipore, Billerica, MA, USA).
Other reagent solutions, such as ammonium acetate and acetic
acid, were of analytical grade (Sino Pharm Chemical Reagent Co.,
Ltd., Shanghai, China). Chemical standards of thymine, thymidine,
2 -deoxyuridine, adenosine, 2 -deoxyinosine, xanthine, inosine,
2 -deoxyguanosine, cytidine, guanosine, guanosine 3 ,5 -cyclic
monophosphate (cGMP), 2 -deoxyadenosine-5 -monophosphate
and cytidine-5 -monophosphate (CMP) were from Sigma Chemical
Co. (St. Louis, MO, USA). Chemical standards of adenine, hypoxan-
thine, uridine, guanine and adenosine 3 ,5 -cyclic monophosphate
(cAMP) were purchased from the National Institute for the Control
of Pharmaceutical and Biological Products (Beijing, China). Refer-
ence compounds of 2 -deoxyadenosine and 2 -deoxycytidine were
obtained from Aladdin Chemical Co. (Nanjing, China). The purity
of each compound was above 98%, determined by HPLC analysis.
The chemical structures of these reference compounds are shown
in Fig. 1.
The fruits and leaves of Ziziphus jujuba (ZJ), Z. jujuba var. spinosa
(ZS) and Z. mauritiana (ZM) were collected from different habitats
in China, and the detailed information is listed in Table 1. Their
botanical origins were identied by the corresponding author, and
voucher specimens were deposited at the Herbarium in Nanjing
University of Chinese Medicine, China. After collection, the entire
fruits and leaves were dried at 45 C, respectively, which is better
consistent with the current production facts of the Ziziphus plants.
2.2. Preparation of standard solutions
Standard stock solutions (100 g mL1 ) were prepared by dis-
solving about 10 mg of individual compounds in 100 mL of water.
These solutions were diluted with water to a series of appropriate
concentrations and used to construct calibration curves. All these
solutions were stored in the refrigerator at 4 C before analysis and
were ltered through a 0.22 m membrane prior to injection.
2.3. Preparation of sample solutions
The dried fruits of ZJ, ZS and ZM were divided into sarcocarp,
hardcore and seed parts, which were further pulverized to homoge-
neous powder (40 mesh), respectively. Likewise, the leaf powders
of ZJ and ZS were obtained according to the same method. The dried
powder (1.0 g) was weighed accurately into a 50 mL conical ask
with stopper, and 40 mL water was added accurately. After weigh-
ing the lled ask accurately, ultrasonication (40 kHz) was carried
out at room temperature for 30 min, then weighing again, and the
same solvent (water) was added to compensate for the lost weight
during the extraction as needed. After centrifugation (15,000 x g,
10 min), the supernatant was stored at 4 C and ltered through
a 0.22 m polytetrauoroethylene lter before injection into the
UHPLCMS/MS system for analysis.
2.4. HILICUHPLC-TQ-MS analysis
UHPLC was performed by using a Waters ACQUITY UPLC system
(Waters, Milford, MA, USA), equipped with a binary solvent delivery
system and an auto-sampler. Hydrophilic interaction chromato-
graphic separation was performed on an ACQUITY UPLC BEH Amide
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
optimization of the separation method. The mobile phase was com-
posed of A (0.8% acetic acid and 10 mM ammonium acetate in
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:
40%50% A, and the column was equilibrated for 6 min in the initial
conditions. The ow rate of the mobile phase was 0.4 mL min1 , and
the column temperature was maintained at 35 C with a column
temperature oven. Two cycles of strong (20% acetonitrile, 400 L)
and weak (90% acetonitrile, 600 L) solvents were used to wash
the injecting system between injections, and these washing sol-
vents did not transfer to the column. The injection volume was
1 L. The column eluent was directed to the mass spectrometer.
All the analyses were operated by MassLynxTM XS Software.
Mass spectrometry was performed on a Waters Xevo TQ tan-
dem quadrupole mass spectrometer (Micromass MS Technologies,
Manchester, UK) using an ESI source operated in positive ion mode.
The parameters in the source were set as follows: capillary volt-
age 3.0 kV; source temperature 150 C; desolvation temperature
550 C; cone gas ow 50 L h1 ; desolvation gas ow 1000 L h1 . Data
were collected in multiple reaction monitoring (MRM) or select
ion monitoring (SIM) mode by screening parent and daughter ions
simultaneously. The cone voltage and collision energy were opti-
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)

2-deoxyadenosine-5-monophosphate (19) Cytidine-5-monophosphate (20)

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-
ware.
2.5. Validation of the method
The method was validated for linearity, limits of detection and
quantication (LODs and LOQs), precision (inter-day and intra-day
precision), reproducibility, and stability following the Interna-
tional Conference on Harmonization (ICH) guideline [44] and some
reports on determination analysis [31,45,46].
2.5.1. Calibration curves, LOD and LOQ
Calibration curves were constructed from peak areas of the
reference standards versus their concentrations. Each calibration
curve was performed with at least six appropriate concentra-
tions in duplicate. Linearity evaluation of calibration curve was
accomplished by applying the lack-of-t test using SPSS 16.0 soft-
ware (SPSS, Chicago, IL). The LOD and LOQ for each analyte were
determined at the signal-to-noise ratio (S/N) of about 3 and 10,
respectively.
2.5.2. Precision, repeatability and stability
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
day; while in inter-day variability test, the solutions were examined
in duplicates for consecutive three days. To conrm the repeatabil-
ity, the same weight of ZJF1, ZJS1, ZJH1 and ZJL1 were mixed to
generate the quality control sample (QC sample), and by using the
QC sample, the six sample solutions were independently prepared
and analyzed. One of the QC sample solutions was stored at 20 C
and analyzed at 0, 2, 4, 8, 12, and 24 h, respectively to evaluate their
stability. All these variations were expressed by relative standard
deviation (RSD).
2.5.3. Recovery
A recovery test was applied to evaluate the accuracy of this
method. It was performed by adding the corresponding marker
compounds with three different levels (high, middle and low) to
0.5 g of QC sample which had previously been analyzed in the
repeatability test and the average content determined in repeat-
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.

Sample no. Species Plant part Collection region Collection time

ZJF1 Ziziphus jujuba Sarcocarp Zhongning, Ningxia Sep, 2011

ZJF2 Ziziphus jujuba Sarcocarp Lingwu, Ningxia Sep, 2011
ZJF3 Ziziphus jujuba Sarcocarp Zhongwei, Ningxia Sep, 2011
ZJF4 Ziziphus jujuba Sarcocarp Wuhu, Anhui Aug, 2011
ZJF5 Ziziphus jujuba Sarcocarp Zhanhua, Shandong Oct, 2011
ZJF6 Ziziphus jujuba Sarcocarp Chiping, Shandong Sep, 2011
ZSF1 Ziziphus jujuba var. spinosa Sarcocarp Helan, Ningxia Sep, 2011
ZSF2 Ziziphus jujuba var. spinosa Sarcocarp Fuping, Hebei Sep, 2011
ZSF3 Ziziphus jujuba var. spinosa Sarcocarp Zibo, Shandong Sep, 2011
ZSF4 Ziziphus jujuba var. spinosa Sarcocarp Hetian, Xinjiang Sep, 2011
ZMF1 Ziziphus mauritiana Sarcocarp Yuanmou, Yunnan Feb, 2011
ZMF2 Ziziphus mauritiana Sarcocarp Haikou, Hainan Feb, 2011
ZMF3 Ziziphus mauritiana Sarcocarp Zhanjiang, Guangdong Feb, 2011
ZJS1 Ziziphus jujuba Seed Wuhu, Anhui Aug, 2011
ZSS1 Ziziphus jujuba var. spinosa Seed Helan, Ningxia Sep, 2011
ZSS2 Ziziphus jujuba var. spinosa Seed Fuping, Hebei Sep, 2011
ZSS3 Ziziphus jujuba var. spinosa Seed Zibo, Shandong Sep, 2011
ZSS4 Ziziphus jujuba var. spinosa Seed Hetian, Xinjiang Sep, 2011
ZMS1 Ziziphus mauritiana Seed Yuanmou, Yunnan Feb, 2011
ZMS2 Ziziphus mauritiana Seed Haikou, Hainan Feb, 2011
ZMS3 Ziziphus mauritiana Seed Zhanjiang, Guangdong Feb, 2011
ZJH1 Ziziphus jujuba Hardcore Wuhu, Anhui Aug, 2011
ZSH1 Ziziphus jujuba var. spinosa Hardcore Helan, Ningxia Sep, 2011
ZSH2 Ziziphus jujuba var. spinosa Hardcore Fuping, Hebei Sep, 2011
ZSH3 Ziziphus jujuba var. spinosa Hardcore Zibo, Shandong Sep, 2011
ZSH4 Ziziphus jujuba var. spinosa Hardcore Hetian, Xinjiang Sep, 2011
ZJL1 Ziziphus jujuba Leaf Zhongning, Ningxia Sep, 2011
ZJL2 Ziziphus jujuba Leaf Lingwu, Ningxia Sep, 2011
ZJL3 Ziziphus jujuba Leaf Zhongwei, Ningxia Sep, 2011
ZJL4 Ziziphus jujuba Leaf Wuhu, Anhui Aug, 2011
ZSL1 Ziziphus jujuba var. spinosa Leaf Helan, Ningxia Sep, 2011
ZSL2 Ziziphus jujuba var. spinosa Leaf Fuping, Hebei Sep, 2011
ZSL3 Ziziphus jujuba var. spinosa Leaf Zibo, Shandong Sep, 2011
ZSL4 Ziziphus jujuba var. spinosa Leaf Hetian, Xinjiang Sep, 2011

for the recovery test. The spiked samples were then extracted,
processed, and quantied in accordance with the methods men-
tioned above, and triplicate experiments were performed on
each level. The average recoveries were estimated by the for-
mula: recovery (%) = [(amount found original amount)/amount
added] x 100%.
2.5.4. Matrix effect
The matrix effect was dened as the ion suppression or enhance-
ment of the ionization of analytes. Because it is very difcult to
nd blank matrix samples free of nucleobases, nucleosides and
nucleotides, the slope comparison method was used to evaluate
the matrix effect for this study [47,48]. The sample extracts, which
were spiked with appropriate amounts of standards as done for the
apparent recovery measurement, were used to construct standard
addition calibration curves. Afterwards, the slopes of the calibration
curves from the standard addition experiments were compared
with the slopes obtained from the pure aqueous standards on the
same concentration levels. The slope ratios (slope matrix/slope sol-
vent) of 1 indicates that matrix does not signicantly suppress or
enhance the response of MS, otherwise denoting ionization sup-
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)

1 Thymine 127.05 127.0 20 1.45

2 Thymidine 243.10 243.1 127.0 10 10 1.68
3 2 -Deoxyuridine 229.08 229.0 112.9 8 10 1.90
4 2 -Deoxyadenosine 252.11 252.0 135.9 16 24 2.26
5 Adenine 136.06 136.0 20 2.51
6 Hypoxanthine 137.05 137.0 16 2.70
7 Uridine 245.08 245.1 113.0 10 10 2.71
8 Adenosine 268.10 268.1 136.0 22 18 2.91
9 2 -Deoxyinosine 253.09 253.0136.9 22 12 3.10
10 Xanthine 153.04 153.0 16 3.11
11 2 -Deoxycytidine 228.10 228.0 111.9 28 10 3.73
12 Inosine 269.09 269.0 136.9 10 14 4.02
13 Guanine 152.06 152.0 16 4.51
14 2 -Deoxyguanosine 268.10 268.1 152.0 10 12 4.81
15 Cytidine 244.09 244.0 112.00 28 10 5.12
16 Guanosine 284.10 284.1 152.0 14 14 6.81
17 cAMP 330.06 330.0 136.0 36 26 8.32
18 cGMP 346.06 346.0 152.0 40 20 8.69
19 2 -Deoxyadenosine-5 -monophosphate 332.08 332.0 135.9 20 16 8.94
20 CMP 324.06 324.0 111.9 16 14 9.37
 S. Guo et al. / J. Chromatogr. A 1301 (2013) 147155
2.6. Data processing and statistical analysis
Data were processed using the TargetLynx application manager
for the quantication of compounds. Principle component analysis
(PCA) was performed using SPSS 16.0 software.
3. Results and discussion
3.1. Optimization of UHPLC conditions
Due to the high polarity, these compounds analyzed in this
study had poor retention property on reversed-phase column,
especially the nucleotides, which did not have any retention even
if the aqueous mobile phases without organic solvent were used.
It was therefore necessary to apply alternative stationary phases.
We employed the HILIC column for separation of these polar com-
pounds and the separation conditions were investigated. Firstly,
two types of HILIC column, a BEH HILIC column (2.1 mm x 100 mm,
1.7 m) and a BEH Amide column (2.1 mm x 100 mm, 1.7 m) were
compared. The results (Fig. S1) showed that the latter had bet-
ter resolution for these hydrophilic components than the former
with the same mobile phase. Secondly, different mobile phase addi-
tives, except for those incompatible with ESI source such as ion
pairing agents or non-volatile buffer salts, were compared. The
results (Fig. S2) showed that ammonium acetate used as a salt
additive of mobile phase could provide much improved resolution
for these compounds compared to ammonium formate. However,
peak tailings were observed for most of target peaks, especially
for those monophosphate compounds such as 2 -deoxyadenosine-
5 -monophosphate and cytidine-5 -monophosphate, when only
ammonium acetate was used as a mobile phase modier. To solve
the above problem, acetic acid was selected and added in the mobile
phase, and improved peak shapes were observed with the increase
of the acetic acid concentration of the eluents. Considering the facts
that the solubility of the acetic acid in solvent B (acetonitrile) is low
and higher concentration of this additive could decrease the sen-
sitivity of the MS, the modier concentrations of the eluents were
determined as solvent A (0.8% acetic acid and 10 mM ammonium
acetate in aqueous solution) and B (0.1% acetic acid in acetonitrile),
and under this condition, all the retention times and peak shapes
were acceptable. Besides, even with specic MS/MS detection, the
baseline separation should be evidently obtained for some ana-
lytes with similar MS/MS characteristics. Such problematic analytes
are cytidine and uridine, 2 -deoxycytidine and 2 -deoxyuridine, as
well as adenine and hypoxanthine. For separating the above ana-
lytes, the elution procedure was optimized as the gradient elution:
06 min, 10% A; 69 min, 10%40% A; 912 min: 40%50% A. The
results showed that the optimized elution procedure mentioned
above could fully separate these problematic compounds, as well
as the others within 10 min. Moreover, since the polarities of the
analytes tested in this study are very strong, different analyte con-
centrations dissolved in water were prepared for standard and
sample solutions. However, water could inuence the peak shape
when the injection volume was large in HILIC mode. Thus, as a com-
promise, 1 L was selected as the injection volume in this study.
The typical chromatograms of the target compounds with MRM
and SIM mode are presented in Fig. 2. Additionally, it was found
the Amide column should be equilibrated for longer time than C18
column, and failure to appropriately equilibrate the column could
result in drifting retention times. In this study we found that the
column was equilibrated for 6 min, the phenomenon of drifting
retention times disappeared. So the equilibration time was set as
6 min.
Recently, studies have been reported UHPLC approach for anal-
ysis of nucleobases and nucleosides using HILIC coupled with the
151
tandem mass spectrometer [49,50], while few for simultaneous
separation of nucleotides, nucleobases and nucleosides in a single
run, like presented in this study.
3.2. Optimization of TQ-MS conditions
To optimize the MS conditions for each analyte, the target
compounds in this study were rstly detected by direct full scan
mass spectrometry method in both positive and negative ioniza-
tion modes. The results (Table S1) showed that the sensitivity and
intensity of analytes signals obtained from the positive ion mode
were higher than those from the negative ion mode. Therefore, the
ESI+ mode was selected in the following studies. In our preliminary
test, the simple MS mode was used for analyzing these com-
pounds, while the results showed that mutual interference may
happen between the compounds producing ions with similar m/z.
For example, both guanine and 2 -deoxyguanosine could provide
the ion at m/z 152.0, and their retention behaviors are similar, thus
these compounds were difcult to distinguish when using simple
MS mode. To minimize this problem and improve the sensitivity of
the analytes, we chose MRM mode in this study. In this way, the
peak could appear only when the appropriate precursor and prod-
uct ions were both detected by selecting the proper ion transitions.
To select a proper transition for the MS/MS detection of the ana-
lyte, all the compounds were examined separately in direct infusion
mode and at least two precursor/product ion pairs for each analyte
were provided. And then, according to the quantitative results, the
highest sensitive and specic ion pairs were selected for the MRM
determination. Once the most appropriate precursor/product ion
pairs had been determined, the values of cone voltage and colli-
sion energy were optimized using the IntelliStart software. For all
the compounds analyzed in this study, the protonated molecule
[M+H]+ were detected with the abundant intensities, and were
selected as the precursor ions. For the product ions, the ribonu-
cleosides including adenosine, guanosine, cytidine, uridine, and
inosine could produce the abundant fragments of [M+H 132]+ ,
which corresponds to the neutral loss of a ribose moiety. Simi-
larly, the fragments of [M+H 116]+ , the ions loss of ribose moiety,
were selected as the product ions for the deoxynucleosides (thymi-
dine, 2 -deoxyadenosine, 2 -deoxyguanosine, 2 -deoxycytidine,
2 -deoxyuridine, 2 -deoxyinosine). For the compounds containing
monophosphate moieties (2 -deoxyadenosine-5 -monophosphate
and cytidine-5 -monophosphate) and 3 ,5 -cyclic monophosphate
moieties (cAMP and cGMP), their corresponding base fragments
exhibited strong signals, so these ions were selected as the prod-
uct ions for detection. Since the abundance of product ions for
nucleobases are too low to be detected by MRM, SIM was used for
their quantitative determination. In addition, we also tried to detect
uracil, however, it was observed that its sensitivity in mass detec-
tion was fairly low, which could be ascribed to the fact that uracil
is lack of basic nitrogens [9]. Thereby, uracil was not determined in
this study. All the MRM transitions and parameters applied in the
study are listed in Table 2.
3.3. Method validation
The proposed HILICUHPLC-TQ-MS method for quantitation of
nucleotides, nucleosides and nucleobases was validated to deter-
mine the linearity, LOD, LOQ, intra-day and inter-day precisions,
stability, accuracy and matrix effect. The results are summarized
in Tables 3 and 4. The correlation coefcient values (r2 ) were bet-
ter than 0.9921 for all analytes. Linearity evaluation of calibration
curve was accomplished by applying the lack-of-t test. As a result,
signicance values greater than 0.05 were obtained for all ana-
lytes at 95% condence level, indicating good correlations between
the investigated compounds concentrations and their peak area
152 S. Guo et al. / J. Chromatogr. A 1301 (2013) 147155

within the test ranges. Since the eluent of the proposed HILIC is
mainly the aqueous organic solution which enhances the analyte
ionization with ESI process, the method showed high sensitivity
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
the IP-RP-LC method [31] and also lower than those obtained
with the method using the same column while detected by Q-
Tof MS detector [49]. The overall intra- and inter-day variations
(RSDs) based on the peak areas of the analyte were in the range of
0.63%4.84% and 1.56%6.37%, respectively. The repeatability and
stability present as RSD were from 0.93% to 7.14% and 2.44% to
6.28%. The overall recoveries were between 90.1% and 103.1% with
RSD between 2.23% and 6.32%. The slope ratio values of matrix
curve to neat solution curve were between 0.90 and 1.06, indi-
cating that the matrix effect on the ionization of analytes was
not obvious under these conditions. These results revealed that
the developed HILICUHPLC-TQ-MS method was sensitive, repeat-
able, and accurate for the quantication analysis of these target
compounds.
3.4. Application to the analysis of real samples
In order to show the utility of the method in proling studies
of nucleotides, nucleosides and nucleobases, an application in real
samples was performed. Total 34 batches of samples from three
Ziziphus plants (ZJ, ZS and ZM) with different parts including sar-
cocarp, seed, hardcore and leaf were analyzed with the established
HILICUHPLC-TQ-MS method. For the seed sample of ZJ, only the
sample from Wuhu, Anhui province was analyzed because no seeds
were found in other samples. Table S2 shows the concentrations of
the individual as well as the total contents of these compounds ana-
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

10 0 thymidine: 243.1> 127.0 10 0 inosine: 269.0> 136.9

2 8.59e4 12 5.02e5
% %

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 2-deoxyuridine: 229.0> 112.9 10 0

3 2.59e4 13 guanine: 152.0 > 152.0
1.02e5
% %

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 2-deoxyadenosine: 252.0> 135.9 10 0 2-deoxyguanosine: 268.1> 152.0

4 9.63e5
%
14 2.02e5
%

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 5 adenine: 136.0> 136.0 10 0 cytidine: 244.0> 112.0

%
2.90e5 %
15 1.94e5

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 guanosine: 284.1> 152.0

6 hypoxanthine: 137.0> 137.0
4.51e5
16 2.16e5
% %

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 cAMP: 330.0> 136.0

uridine: 245.1> 113.0
7 1.60e5 17 4.04e4
% %

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 adenosine: 268.1> 136.0 10 0

8 2.32e6 18 cGMP: 346.0> 152.0
1.98e4
% %

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

10 0 thymidine: 243.1> 127.0 10 0

2 9.34e4 12 inosine: 269.0> 136.9
1.70e5
% %

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 guanine: 152.0> 152.0

10 0
3 2-deoxyuridine: 229.0> 112.9
2.59e4
13 1.94e5
% %

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

10 0 uridine: 245.1> 113.0 10 0

7 1.96e5 17 cAMP: 330.0> 136.0
2.59e6
% %

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

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 )

1 Thymine y = 4992x + 468 0.9938 0.0312.496 3.12 12.48

2 Thymidine y = 46,081x + 479 0.9986 0.0103.936 0.66 1.97
3 2 -Deoxyuridine y = 8026x + 153 0.9997 0.0142.740 1.70 6.85
4 2 -Deoxyadenosine y = 1,033,460 x + 22,716 0.9961 0.0021.220 0.12 0.31
5 Adenine y = 352,639x + 9443 0.9971 0.0021.216 0.31 0.93
6 Hypoxanthine y = 316,715x + 25,155 0.9940 0.0041.808 0.45 1.51
7 Uridine y = 50,729x + 6424 0.9921 0.0173.376 0.84 2.52
8 Adenosine y = 1,762,738 x + 23,264 0.9979 0.0021.152 0.11 0.29
9 2 -Deoxyinosine y = 54,376x 245 0.9986 0.0142.800 0.88 3.50
10 Xanthine y = 42,729x + 166 0.9957 0.0102.080 1.25 5.02
11 2 -Deoxycytidine y = 301,946x 14,491 0.9978 0.0062.925 0.59 2.36
12 Inosine y = 408,201x + 14,000 0.9983 0.0052.256 0.14 0.56
13 Guanine y = 89,427x + 3645 0.9948 0.0052.640 0.68 2.64
14 2 -Deoxyguanosine y = 328,158x 106 0.9966 0.0031.280 0.31 0.64
15 Cytidine y = 122,673x 3251 0.9968 0.0051.144 0.57 2.28
16 Guanosine y = 467,665x + 2587 0.9997 0.0031.474 0.12 0.37
17 cAMP y = 47,838x + 1209 0.9994 0.0189.120 0.59 2.36
18 cGMP y = 50,916x + 407 0.9983 0.0062.800 0.70 2.80
19 2 -Deoxyadenosine-5 -monophosphate y = 29,342x 361 0.9993 0.0083.925 1.57 6.28
20 CMP y = 32,820x 301 0.9995 0.02003.900 2.44 9.75
a
y is the value of peak area, and x is the value of the reference compounds concentration (g mL1 ).
154 S. Guo et al. / J. Chromatogr. A 1301 (2013) 147155

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)

Intraday (n = 6) Interday (n = 6) Mean RSD, %

1 Thymine 2.9 6.3 4.5 4.3 91.4 4.1 0.90

2 Thymidine 1.6 2.7 3.2 3.1 93.4 3.3 0.91
3 2 -Deoxyuridine 4.8 4.2 7.1 5.0 96.4 3.2 0.95
4 2 -Deoxyadenosine 1.1 2.6 1.7 3.6 99.1 2.8 1.04
5 Adenine 0.6 4.2 1.0 4.2 95.5 4.1 0.93
6 Hypoxanthine 0.7 3.0 1.9 2.9 93.3 4.0 0.97
7 Uridine 1.1 5.1 1.4 4.1 91.6 5.1 0.92
8 Adenosine 0.7 2.0 2.5 3.1 97.2 2.2 0.96
9 2 -Deoxyinosine 2.5 4.8 3.0 5.3 94.5 3.8 0.95
10 Xanthine 3.1 6.4 4.4 6.3 94.1 5.6 0.90
11 2 -Deoxycytidine 0.8 1.6 5.0 3.1 95.6 2.7 0.94
12 Inosine 0.8 3.9 0.9 2.4 97.9 1. 6 0.96
13 Guanine 3.8 4.9 2.3 5.3 94.3 4.3 0.95
14 2 -Deoxyguanosine 1.0 5.2 5.7 2.6 100.5 5. 8 1.05
15 Cytidine 0.9 3.7 1.8 3.0 93.1 4.3 0.94
16 Guanosine 1.0 2.6 2.1 2.9 103.1 4.2 1.03
17 cAMP 1.1 4.1 5.5 4.3 94.5 2.7 1.06
18 cGMP 2.6 3.9 6.7 3.2 96.7 4.6 1.04
19 2 -Deoxyadenosine-5 -monophosphate 3.3 6.0 3.1 5.0 90.1 3.4 0.91
20 CMP 3.8 6.4 5.3 5.3 94.7 6.3 0.90
a
Matrix effects are calculated by slope matrix/slope solvent.

hardcore parts (ZJH1 and ZSH14) were rich in nucleobases, nucle-
osides and nucleotides, especially those from leaf parts (ZJL14 and
ZSL14) which have not been researched or utilized until now. The
above ndings suggested the leaves of these Ziziphus plants could
be selected as the promising natural sources for future industrial
research of nucleosides, nucleobases and nucleotides. As for the
different species, the contents of these analytes in sarcocarp of ZS
were lower than those of ZJ and ZM. As for the individual com-
pound determined in the experiments, the remarkable differences
were also observed. For example, the contents of ribonucleosides
in different parts of Ziziphus plants were obviously larger than
those of deoxynucleosides, and the latter was found mainly in the
leaves. As for the nucleotides, it was found that CMP mainly exist in
the seeds, while 2 -deoxyadenosine-5 -monophosphate was only
detected in trace amount in the samples of leaf and some fruits. Fur-
thermore, it was determined that the cyclic nucleotides including
cAMP and cGMP are the predominant constituents in the sarcocarp
samples and the highest content were of 984 g g1 and 675 g g1 ,
respectively, while they could not be detected in the samples from
other parts.
3.5. PCA of the samples
To evaluate the variation between the different parts of these
Ziziphus plants, PCA was performed on the basis of the contents
of 20 tested compounds from UHPLC proles. The rst three prin-
cipal components (PC 1, PC 2 and PC 3) with >62% of the whole
variance, were extracted for analysis. The remaining principal com-
ponents, which had a minor effect on the model, were discarded.
The components loading matrix is shown in Table S3. According to
their loadings, most of the analytes showed good correlation with
the rst three principal components. The sample scatter plots are
shown in Fig. 3, where each sample is represented as a marker. It
was noticeable that the samples were clustered into two domains
(seed vs sarcocarp, leaf vs sarcocarp, seed vs leaf), respectively.
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
Ziziphus plants have various chemical proles of the nucleosides,
nucleobases and nucleotides, which could be selected as the chem-
ical markers for these samples quality control.
4. Conclusions
In this study, a reliable, simple, and sensitive method capa-
ble of quantifying 20 nucleotides, nucleosides and nucleobases in
Ziziphus plants was established by using an HILICUHPLC-TQ-MS
method. By combination of HILIC and UHPLC approach, the more
polar compounds, especially the nucleotides, were sufciently sep-
arated within 10 min without utilization of the ion pairing reagents
which were not suggested for ESI source due to the low volatility
or decreasing the sensitivity of the ESI-MS detector. The analy-
sis results showed that the fruits and leaves of Ziziphus plants are
rich in nucleosides and nucleobases, and could be selected as the
healthy foods. Our results in the present study clearly suggested
that HILICUHPLC-TQ-MS method could be employed as a useful
tool for quality assessment of the samples from the Ziziphus plants
using nucleotides, nucleosides and nucleobases as the markers. Fur-
thermore, this validated method could be valuable for the routine
quantitation of nucleotides, nucleosides and nucleobases from food
and plant samples.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 30672678), and the Youth Natural Sci-
ence Foundation of Nanjing University of Chinese Medicine (No.
12XZR01), the Priority Academic Program Development of Jiangsu
Higher Education Institutions (ysxk-2010), 2009 Program for New
Century Excellent Talents by the Ministry of Education (NCET-
09-0163). We are grateful to Mr. Decang Kong (Cangxian, Hebei
province, China) and M.D. Pengfei Hou for collecting samples. We
are also pleased to thank Waters China Ltd. for technical support.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.chroma.2013.05.074.
References
[1] K.A. Jacobson, M.F. Jarvis, M. Williams, J. Med. Chem. 45 (2002) 40574093.
[2] V. Ralevic, G. Burnstock, Pharmacol. Rev. 50 (1998) 413492.
[3] G. Anfossi, I. Russo, P. Massucco, L. Mattiello, F. Cavalot, A. Balbo, M. Trovati,
Thromb. Res. 105 (2002) 7178.
[4] J.B. Conti, L. Belardinelli, D.B. Utterback, A.B. Curtis, Circulation 91 (1995)
17611767.
[5] L. Virag, C. Szabo, FASEB J. 15 (2001) 99107.
[6] A.P. Schmidt, D.R. Lara, J. de Faria Maraschin, A. da Silveira Perla, D. Onofre
Souza, Brain Res. 864 (2000) 4043.
[7] J.J. Kinahan, E.P. Kowal, G.B. Grindey, Cancer Res. 41 (1981) 445451.
[8] F.Q. Yang, S.P. Li, J. Pharm. Biomed. Anal. 48 (2008) 231235.
155
[9] J.L. Gao, K.S.Y. Leung, Y.T. Wang, C.M. Lai, S.P. Li, L.E. Hu, G.H. Lu, Z.H. Jiang, Z.L.
Yu, J. Pharm. Biomed. Anal. 44 (2007) 807811.
[10] P. Chen, W. Li, Q. Li, Y.H. Wang, Z.G. Li, Y.F. Ni, K. Koike, Talanta 85 (2011)
16341641.
[11] Z.M. Qian, J.B. Wan, Q.W. Zhang, S.P. Li, J. Pharm. Biomed. Anal. 48 (2008)
13611367.
[12] B.Z. Duan, L.Z. Wang, X.H. Dai, L.F. Huang, M.R. Yang, S.L. Chen, Anal. Lett. 44
(2011) 24912502.
[13] R. Liu, J. Ji, L.C. Wang, S.Y. Chen, S. Guo, H. Wu, Food Chem. 135 (2012) 548554.
[14] A. Ranogajec, S. Beluhan, Z. Smit, J. Sep. Sci. 33 (2010) 10241033.
[15] S. Guo, J.A. Duan, Y.P. Tang, Z.H. Zhu, Y.F. Qian, N.Y. Yang, E.X. Shang, D.W. Qian,
J. Agric. Food Chem. 58 (2010) 1077410780.
[16] L. Yu, J. Zhao, S.P. Li, H. Fan, M. Hong, Y.T. Wang, Q. Zhu, J. Sep. Sci. 29 (2006)
953958.
[17] J.H. Zhou, X. Xu, L.P. Sun, Y. Li, L. Huang, Anal. Methods 4 (2012) 37923797.
[18] J. Czarnecka, M. Cieslak, K. Michal, J. Chromatogr. B 822 (2005) 8590.
[19] H. Fan, S.P. Li, J.J. Xiang, C.M. Lai, F.Q. Yang, J.L. Gao, Y.T. Wang, Anal. Chim. Acta
567 (2006) 218228.
[20] S. Cohen, M. Megherbi, L.P. Jordheim, I. Lefebvre, C. Perigaud, C. Dumontet, J.
Guitton, J. Chromatogr. B 877 (2009) 38313840.
[21] H.Y. Li, S.M. Wang, H.M. Liu, S.S. Bu, J. Li, D. Han, M.Z. Zhang, G.Y. Wu, J. Mass
Spectrom. 44 (2009) 641651.
[22] P. Liu, Y.Y. Li, H.M. Li, D.J. Wan, Y.J. Tang, Anal. Chim. Acta 687 (2011) 159167.
[23] F.Q. Yang, J. Guan, S.P. Li, Talanta 73 (2007) 269273.
[24] S. Studzinska, B. Buszewski, J. Chromatogr. B 887 (2012) 93101.
[25] H.Y. Cheung, C.W. Ng, D.J. Hood, J. Chromatogr. A 911 (2001) 119126.
[26] X.J. Chen, F.Q. Yang, Y.T. Wang, S.P. Li, Electrophoresis 31 (2010) 20922105.
[27] F.Q. Yang, S.P. Li, P. Li, Y.T. Wang, Electrophoresis 28 (2007) 16811688.
[28] Y.Q. Jiang, Y.F. Ma, Anal. Chem. 81 (2009) 64746480.
[29] F.Q. Yang, L.Y. Ge, J.W.H. Yong, S.N. Tan, S.P. Li, J. Pharm. Biomed. Anal. 50 (2009)
307314.
[30] E. Fromentin, C. Gavegnano, A. Obikhod, R.F. Schinazi, Anal. Chem. 82 (2010)
19821989.
[31] F.Q. Yang, D.Q. Li, K. Feng, D.J. Hu, S.P. Li, J. Chromatogr. A 1217 (2010)
55015510.
[32] J. Jia, H. Zhang, L.A. Zhao, Z.Y. Zhu, G.Q. Zhang, Y.F. Chai, Chromatographia 73
(2011) 755759.
[33] P. Jandera, Anal. Chim. Acta 692 (2011) 125.
[34] D.C.A. Neville, D.S. Alonzi, T.D. Butters, J. Chromatogr. A 1233 (2012) 6670.
[35] B. Buszewski, S. Noga, Anal. Bioanal. Chem. 402 (2012) 231247.
[36] J. Bernal, A.M. Ares, J. Pol, S.K. Wiedmer, J. Chromatogr. A 1218 (2011)
74387452.
[37] T. Langrock, P. Czihal, R. Hoffmann, Amino Acids 30 (2006) 291297.
[38] S. Guo, J.A. Duan, D. Qian, Y. Tang, Y. Qian, D. Wu, S. Su, E. Shang, J. Agric. Food
Chem. 61 (2013) 27092719.
[39] Y. Chen, W.F. Bicker, J.Y. Wu, M.Y. Xie, W.G. Lindner, J. Agric. Food Chem. 60
(2012) 42434252.
[40] R. Tuytten, F. Lemiere, W. Van Dongen, E. Witters, E.L. Esmans, R.P. Newton, E.
Dudley, Anal. Chem. 80 (2008) 12631271.
[41] E. Johnsen, S.R. Wilson, I. Odsbu, A. Krapp, H. Malerod, K. Skarstad, E. Lundanes,
J. Chromatogr. A 1218 (2011) 59815986.
[42] G.S. Philibert, S.V. Olesik, J. Chromatogr. A 1218 (2011) 82228230.
[43] X. Cai, L. Zou, J. Dong, L. Zhao, Y. Wang, Q. Xu, X. Xue, X. Zhang, X. Liang, Anal.
Chim. Acta 650 (2009) 1015.
[44] ICH, Guidance for Industry, Q2B Validation of Analytical Procedures: Method-
ology, ICH, Rockville, 1996.
[45] Y. Zhou, S. Lee, F.F.K. Choi, G. Xu, X. Liu, J.Z. Song, S.L. Li, C.F. Qiao, H.X. Xu, Anal.
Chim. Acta 678 (2010) 96107.
[46] Z.X. Yan, X.H. Yang, J.B. Wu, H. Su, C. Chen, Y. Chen, Anal. Chim. Acta 691 (2011)
110118.
[47] K. Granby, J.H. Andersen, H.B. Christensen, Anal. Chim. Acta 520 (2004)
165176.
[48] L. Chen, F. Song, Z. Liu, Z. Zheng, J. Xing, S. Liu, J. Chromatogr. A 1225 (2012)
132140.
[49] G. Paglia, S. Hrafnsdttir, M. Magnsdttir, R.M.T. Fleming, S. Thorlacius, B..
Palsson, I. Thiele, Anal. Bioanal. Chem. 402 (2012) 11831198.
[50] Z. Yan, J. Sun, J. Wang, Y. Xu, Y. Chang, P. Meng, M. Zhu, Q. Fu, Y. Sun, Z. He, J.
Chromatogr. B 878 (2010) 466470.