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794
ISSN 0326-2383
KEY WORDS: Human plasma, HPLC, Validation, Citicoline monosodium.
* Author to whom correspondence should be addressed. E-mail: prof_piyushtrivedi@yahoo.com, piyush.trivedi@rgtu.net
Latin American Journal of Pharmacy
(formerly Acta Farmacéutica Bonaerense)
Lat. Am. J. Pharm. 30 (4): 794-8 (2011)
Short communication
Received: August 25, 2010
Revised version: September 8, 2010
Accepted: September 13, 2010
Development and Validation of a RP-HPLC Method for
Determination of Citicoline Monosodium in Human Plasma
Shailendra K. BINDAIYA, Kapendra SAHU, Mukesh BHAISARE, C. KARTHIKEYAN,
N.S.H.N. MOORTHY, Farhad F. MEHTA & Piyush TRIVEDI*
School of Pharmaceutical Sciences, Rajiv Gandhi Proudyogiki Vishwavidyalaya,
Bhopal-462036, India
SUMMARY. A sensitive and specific high performance reversed phase liquid chromatographic method
was developed for quantification of citicoline monosodium (CTM) in human plasma. The active drug was
isocratically eluted at a flow rate of 1 ml/min at ambient temperature in a nucleosil C18 analytical column
with a mobile phase composed of tetrabutyl ammonium hydrogen sulfate buffer (0.005 M, pH
5.0):methanol (95:05, v/v). Photodiode array (PDA) was performed at 270 nm and the retention time of the
drug was found to be 6.64 min. The lowest limit of quantification (LLOQ) and of detection (LOD) were
found to be 30 and 10 ng/ml, respectively. The method was validated and the response was found to be lin-
ear in the drug (CTM in spiked plasma) concentration range 150-900 ng/ml. The method was found to be
accurate, with ranging from 96.38 to 98.65 % and precise, with intra-day, inter-day as well as analyst-to-
analyst precision. The total recoveries of the method ranged between 95.69 and 97.89 %. Stability data re-
vealed that the drug is stable in human plasma under various test conditions and the method can be suc-
cessfully used for analysis of CTM in human plasma and in pharmacokinetic studies.
INTRODUCTION
Citicoline monosodium (CTM), also known
as cytidine diphosphate-choline (CDP-choline)
and cytidine 5’-diphosphocholine is a psychos-
timulant/nootropic. CTM is chemically 5’-O-[hy-
droxyl ({hydroxyl [2-(trimethylammonio)ethoxy]
phosphoryl}oxy) phosphoryl]cytidine (Fig. 1).
Citicoline is a complex organic molecule that
functions as an intermediate in the biosynthesis
of cell membrane phospholipids. Exogenous
citicoline research in animal experiments and
human clinical trials provides evidence of its
cholinergic and neuroprotective actions. As a di-
etary supplement, citicoline appears useful for
improving both the structural integrity and func-
tionality of the neuronal membrane that may as-
sist in membrane repair. Citicoline has been
shown to improve memory and other cognitive
functions in patients with chronic cerebrovascu-
lar disease or dementia and in old people suf-
fering from memory deficit without dementia
1,2
.
It also improves memory, behaviour and the
overall clinical impression in old people suffer-
ing from chronic brain diseases like Alzheimer’s.
A survey of literature reveals no reports on ana-
lytical methods for quantification of CTM in hu-
man plasma. Hence, the objective of the present
research work is to develop a simple, rapid,
sensitive and reproducible method for analysis
of CTM in plasma and perform its validation ac-
cording to ICH and FDA guidelines
3-5
. To the
Figure 1. Structure of citicoline monosodium.
795
Latin American Journal of Pharmacy - 30 (4) - 2011
best of our knowledge this is the first such at-
tempt to be recorded in literature.
MATERIAL AND METHODS
Chemicals and reagents
CTM was obtained as gratis sample from
Torrent Pharmaceutical Limited, Ahmedabad, In-
dia. Methanol of HPLC grade was purchased
from Merck Ltd, New Delhi, India. HPLC grade
water (Milli Q Water) was purchased from Ran-
baxy Laboratories Ltd, (New Delhi, India). Hu-
man plasma was obtained from Blood Bank De-
partment, Bhopal Memorial Hospital & Research
Centre, Bhopal, (M.P.), India. All other chemi-
cals of analytical grade were purchased from lo-
cal vendors.
Instrumentation
The HPLC system (Shimadzu, Japan) consist
of a LC-10AT VP pump, a SPD-10AVP, PDA de-
tector, a nucleosil C18 (250 mm X 4.6 mm, 5
µm) column, a Phenomenex, HPLC guard car-
tridge system and a Class LC10/M10A software.
Chromatographic conditions
Chromatographic analysis was performed at
ambient temperature on a nucleosil C18 analyti-
cal column with a mobile phase composed of
tetrabutyl ammonium hydrogen sulfate buffer
(1.69 g dissolved in 1000 ml, 0.005 molar solu-
tion), pH 5.0, adjusted with diluted acetic acid:
methanol (95:05, v/v). A small sample volume
of 20 µl was used for each sample run, being in-
jected into the HPLC system. The drug was iso-
cratically eluted at a flow rate of 1 ml/min and
the chromatogram was monitored with UV de-
tector at a wavelength of 270 nm.
Preparation of the calibration standards
and quality control (QC) samples
Standard stock solutions were prepared by
dissolving 50 mg of CTM in 50 ml of mobile
phase to obtain final concentrations of 1000
µg/ml (stock-A). From this stock, 5 ml solution
was taken and diluted to 50 ml with mobile
phase to give 100 µg/ml of CTM standard solu-
tion (stock-B). Aliquots of stock-B were further
diluted to obtain different concentrations: 150,
300, 450, 600, 750, and 900 ng/ml. To prepare
standard spike plasma stock solutions, 1 ml was
taken from stock-A, and diluted to 10 ml with
blank plasma to give 100 µg/ml of CTM (stock-
C). From the working standard solution of
stock-C, different concentrations were prepared
by taking 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 ml, and
then the volume was made up, in each case, to
5 ml with blank plasma. For all the concentra-
tions, 5 ml of precipitating agent (methanol)
was added to obtain the final concentrations of
150, 300, 450, 600, 750 and 900 ng/ml for CTM.
QC samples at four different levels such as HQC
(high quality control, 900 ng/ml), MQC (medi-
um quality control, 450 ng/ml), LQC (low quali-
ty control, 150 ng/ml) and LLOQ (lowest limit of
quantitation, 100 ng/ml) of CTM were selected
to perform different validation parameters.
Sample pretreatment and extraction
procedure
Drugs were extracted from plasma samples
using a protein precipitation technique.
Methanol was selected as the precipitating agent
and each plasma sample gave satisfactory values
for recovery with a single extraction. The satis-
factory result was obtained when 1:4 ratio of
plasma and methanol were mixed thoroughly,
vortexed at room temperature and centrifuged
at 5000 rpm for 6 min. The clear supernatant
liquid was decanted, filtered through a 0.22 µm
syringe filter and injected (20 µl) into HPLC sys-
tem.
Limit of detection (LOD) and lowest limit of
quantification (LLOQ)
The limit of detection (LOD) was defined as
the concentration that yields a signal-to-noise ra-
tio of 3. The lowest limit of quantification
(LLOQ) was calculated to be the lowest analyte
concentration that could be measured with a
signal-to-noise ratio of 10. To determine LOD
and LLOQ, plasma samples were spiked with
decreasing concentrations of the analyte and
were analyzed experimentally.
Method Validation
Calibration and linearity
Calibration plot for the analyte in plasma
were prepared by spiking drug-free plasma with
standard stock solutions to yield concentrations
of 150-900 ng/ml. The solutions were injected in
replicates (n = 6) into the HPLC column while
keeping the injection volume constant (20 µl).
Calibration curves were constructed by using ra-
tio of the observed analyte peak area versus
concentration of analyte. Intercept, slope and
correlation coefficient (r
2
) were determined by
linear regression data analysis, which were then
used to calculate the analyte concentration in
each sample.
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BINDAIYA S.K., SAHU K., BHAISARE M., KARTHIKEYAN C., MOORTHY N.S.H.N., MEHTA F.F. & TRIVEDI P.
Precision and accuracy
The inter-day, intra-day, analyst to analyst
precision and accuracy of the assay were deter-
mined by assaying three QC samples and LLOQ
in replicates (n = 6) for each drug. Precision
was expressed as co-efficient of variance (% CV)
and the accuracy was as % nominal concentra-
tion and % bias.
Extraction recovery of CTM from plasma
The extraction recovery of CTM was deter-
mined by calculating the peak areas obtained
from blank plasma samples spiked with analyte
before extraction with those from blank plasma
samples, to which analyte was added after ex-
traction. According to the guidance of USFDA
4
,
recovery experiments should be performed at
three concentrations (low, medium, and high).
So this procedure was repeated for five repli-
cates at three concentrations of 150, 450 and
900 ng/ml as shown in Table 1.
Selectivity
To evaluate the selectivity of the assay, blank
samples of the appropriate biological matrix
(plasma) were prepared from six different
sources.
Stability of the method
Stability of drugs in biological fluids is a
function of the sample storage conditions,
chemical properties of the drug, matrix and the
container system. Blank samples were spiked
with appropriate aliquots of diluted CTM stock
solution to prepare LQC and HQC samples. The
stability of drugs was evaluated under condi-
tions likely to be encountered during actual
sample handling and analysis. These samples
% Accuracy
Measured(ng/ml) % C.V % Recovery
% N.C % Bias
Intra-day
LQC 147.98 98.65 -1.35 1.04 97.89
MQC 433.74 96.38 -3.62 1.32 96.98
HQC 882.91 98.11 -1.89 1.03 96.77
Inter-day
LQC 146.09 97.40 -2.6 1.39 95.69
MQC 434.09 96.46 -3.54 1.26 95.76
HQC 880.62 97.84 -2.16 1.33 96.01
Analyte-to-Analyte
LQC 144.88 96.58 -3.42 1.32 96.34
MQC 435.65 96.81 -3.19 1.41 96.54
HQC 881.33 97.92 -2.08 1.48 95.99
Table 1. Accuracy and precision of the HPLC method for the determination of CTM in spiked plasma samples (n
= 6). n = no. of determination.
Concentration
added (ng/ml)
were kept to evaluate different stability parame-
ters such as stock solution stability (SSS, 6 h at
room temperature), bench top stability (BTS, 12
h at room temperature), post processing stability
(PPS, over the maximum time, i.e., from the
completion of sample work-up to the comple-
tion of data collection), freeze and thaw stability
(FTS, subjected to three freeze–thaw cycles of -
20 °C during 24 h) and long term storage stabili-
ty (LTS, subjected to freeze storage at -20 °C
during the entire period covered by the bioana-
lytical study, i.e., from the first day of sample
preparation to the last day of sample analysis).
For each concentration and storage conditions,
three replicates were analyzed. The concentra-
tion of CTM after each storage period was relat-
ed to the initial concentration as determined for
the samples that were freshly prepared and pro-
cessed immediately.
RESULTS AND DISCUSSION
In the present analysis a simple, precise and
accurate analytical method for the estimation of
CTM in human plasma has been developed and
validated. At the chromatographic conditions se-
lected for this method, the chromatograms for
blank plasma and extracted peaks of CTM at dif-
ferent quality control represents in Figure 2.
The chromatographic behaviour of a blank
plasma and CTM were detected at 270 nm. The
results obtained from the analysis shows that
the ionization and separation with matrix of
CTM were affected by the composition of mo-
bile phase. Therefore, the selection of mobile
phase components as critical and the ammoni-
797
Latin American Journal of Pharmacy - 30 (4) - 2011
um acetate was employed to supply the ionic
strength. With buffers of lower strength, the
peak shapes were not satisfactory, whereas with
higher strength there was an improvement in
the peak shape, however not got satisfactory re-
sults. Finally, the best results were achieved on
the nucleosil C18 column at ambient tempera-
Figure 2. Representative chromatogram (a) for blank
plasma. Extracted chromatogram of citicoline
monosodium (
b) for LQC (c) for MQC (d) for HQC.
S. Drug concentration in Mean
SD %CV %NC
% Relative
No. spiked plasma (ng/ml) peak area error
1. 150 23.717 0.14 0.590 100.531 0.528
2. 300 31.618 0.24 0.759 99.374 -0.630
3. 450 39.489 0.39 0.988 97.171 -2.912
4. 600 47.861 0.46 0.961 97.314 -2.761
5. 750 55.625 0.52 0.935 100.538 0.535
6. 900 63.536 0.61 0.960 101.075 1.063
Table 2. Calibration data for CTM in buffer–methanol (95:05 v/v) after spiking of plasma with stock solutions (n
= 6).
ture using a mobile phase composed of buffer
0.005 M tetrabutyl ammonium hydrogen sulfate,
pH 5.0:methanol 95:05, (v/v), which was run in
a isocratic mode at 1.0 ml/min flow rate. Under
these chromatographic conditions CTM in plas-
ma matrix were resolved with retention time
6.64 min. The method was found to be suitable
because reproducible results were obtained on
each sample run. The validation results obtained
from the analysis showed that the linearity of
the calibration plot for CTM in human plasma at
concentrations range investigated from 150-900
ng/ml was excellent (r
2
=0.999). The linearity re-
sults obtained from the analysis is given in
Table 2.
A typical calibration plot obtained during
plasma analysis could be described by the linear
equation Y = 0.0538X-0.5707, where Y is peak
area and X is concentration (ng/ml). The LOD
and LLOQ for CTM were calculated experimen-
tally. The LOD and LLOQ were found to be 20
ng/ml and 90 ng/ml, respectively. The total ac-
curacy of the method ranged between 96.38 %
and 98.65 %, and the total recoveries ranged
from 95.69 to 97.89 as shown in Table 1. The in-
tra-day precision (%CV) for QC samples (LQC,
MQC and HQC ng/ml) was 1.04 %, 1.32 % and
1.03 %, inter-day analysis was 1.34 %, 1.26 %
and 1.33 %, and that of analyst to analyst was
1.32, 1.41 and 1.48 respectively. The precision
data for the validation studies which gives %CV
values for precision (intra-day and inter-day) is
less than 15 % (as per USFDA bioanalytical
method validation guidelines for industry), indi-
cating that the method is sufficiently precise.
The stability test results (% deviation) for CTM
at LQC, MQC and HQC were as illustrated in
Table 3. The %deviation results are ranged be-
tween 5.786 - 8.673 for LQC, MQC is 2.448 -
5.382 and the HQC is 1.343 - 2.372. The result
of % change demonstrates that stability testing
of CTM is within the acceptance range of ± 15
% deviation from the nominal concentration.
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BINDAIYA S.K., SAHU K., BHAISARE M., KARTHIKEYAN C., MOORTHY N.S.H.N., MEHTA F.F. & TRIVEDI P.
CONCLUSION
A simple, rapid, precise, and rapid reversed
phase HPLC method has been developed and
validated for analysis of CTM in human plasma.
The chromatographic run time is only 6.64 min
makes this method suitable for processing of
many samples in limited time. The method was
validated for analysis of CTM in human plasma
over the range 150 to 900 ng/ml. This assay can
be suitably used for the determination of CTM
in human plasma and should be useful in clini-
cal trials and clinical therapeutic drug monitor-
ing (TDM) programs. It would also be potential-
ly useful in the determination of pharmacokinet-
ic profiles and in bioequivalence studies in psy-
chostimulant research.
Acknowledgements. The authors gratefully acknowl-
edge Vice Chancellor, RGPV for experimental facility
provided for this research work. The authors Shailen-
dra Kumar Bindaiya, Kapendra Sahu, and Mukesh
Bhaisare wish to thank AICTE, New Delhi for post-
Concentration (ng/ml)
LQC (150) MQC (450) HQC (900)
Obs. % Dev. Obs. % Dev. Obs. % Dev.
SSS 141.32 5.786 432.78 3.826 884.23 1.752
BTS 139.46 7.026 438.98 2.448 879.44 2.284
PPS 138.67 7.553 435.99 3.113 887.91 1.343
FTS 136.99 8.673 425.78 5.382 879.98 2.224
LTS 140.12 6.586 427.89 4.913 878.65 2.372
Table 3. Stability of the developed method at various conditions.
Stability
conditions
graduate fellowship. One of the authors C.
Karthikeyan wishes to thank CSIR, New Delhi for
providing Senior Research Fellowship (SRF).
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