European Journal of Medicinal Chemistry 65 (2013) 94e101

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Chromatographic separation of piracetam and its metabolite in a

mixture of microsomal preparations, followed by an MS/MS analysis
Kapendra Sahu a, Anees A. Siddiqui a, *, Mohammad Shaharyar a, Niyaz Ahmad b,
Mohammad Anwar b, Farhan J. Ahmad b
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India
b
Nano Medicine Laboratory, Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi
110062, India

a r t i c l e i n f o a b s t r a c t

Article history: A rapid bioanalytical method was evaluated for the simultaneous determination of piracetam and its
Received 24 November 2012 metabolite (M1) in human microsomal preparations by fast ultra-performance liquid chromatography/
Received in revised form tandem mass spectrometry (UPLCeMS/MS). In addition, a validated method of M1 in rat plasma was
10 March 2013
developed and successfully applied on pharmacokinetic studies. The present study was carried out to
Accepted 26 April 2013
Available online 3 May 2013
determine the metabolic pathways of piracetam for phase I metabolism and used cytochrome P450
isoforms responsible for the piracetam metabolism in human liver microsomes (HLMs). While additional
potential metabolites of piracetam were suggested by computer-modeling. The resulting 2-(2-
Keywords:
Piracetam metabolism
oxopyrrolidin-1-yl) acetic acid was the sole metabolite detected after the microsomal treatment. The
Human liver microsomes amide hydrolysis mainly underwent to form a metabolite i.e., 2-(2-oxopyrrolidin-1-yl) acetic acid (M1).
Metabolic pathway Ó 2013 Elsevier Masson SAS. All rights reserved.
Pharmacokinetics of the generated
metabolite
UPLCeMS/MS
Validation

1. Introduction synthesizing compounds that are more metabolically stable and

A recent trend in the drug discovery process is to shift the
starting point of drug metabolism and pharmacokinetic (DMPK)
studies from late developmental stage to the discovery stage in an
attempt to address potential issues in adsorption, distribution,
metabolism and excretion (ADME), in parallel with the optimiza-
tion of structureeactivity relationship (SAR). In vitro assays such as
metabolic stability, cytochrome P450 (CYP) inhibition, and induc-
tion and cell permeability studies offer a higher throughput than
the corresponding in vivo methods. As such, they have been gaining
popularity as frontline screens to identify potential development
liabilities as well as feedback to medicinal chemists [1,2].
Although not all synthesized compounds could be evaluated in
ADME parameters, including microsomal metabolic stability and
in vivo pharmacokinetics (PK) studies. Knowledge of the metabolic
fate of the compounds is of importance as well because it can
identify parts of the molecule which are prone to metabolism. The
information facilitates to the synthetic chemists for the
* Corresponding author. Tel.: þ91 11 26059688/5650; fax: þ91 11 27048685.
E-mail addresses: kapendra_sahu@yahoo.com (K. Sahu), prof.anees1@gmail.com
(A.A. Siddiqui).
consequently, have a lower clearance rate and a longer half-life [3].
Piracetam (2-oxo-1-pyrolidine acetamide, Fig. 1(a)) which is the
prototype of nootropic drugs is useful for the improvement of
learning and memory and in the treatment of myoclonus [3,4].
Metabolic profiling is a valuable tool to predict the pharmacokinetic
properties of drug candidates and to assess the possibility of druge
drug interaction risks [5,6].
The quadrupole time of flight has proven to be extremely
powerful in quantitative and qualitative drug metabolism studies
because of their sensitivity, selectivity and ease of use. The Q-TOF
has a clear advantage in respect of scan speed and number of MS/MS
experiments that can be performed simultaneously. Moreover, this
instrument allows accurate mass measurements in the MS and MS/
MS scans, which helps data interpretation. Thus, the development of
generic, rapid separation for the identification of metabolites in
microsomal samples by UPLC/MS/MS will be described and address
the influence of several chromatographic and mass spectrometric
parameters to optimize the separation and detection [7e11].
The metabolism of piracetam in human Microsomes and rat
plasma is subjected to hydrolytic metabolism to form the active
carboxylic acid identifiable by UPLC-Q-TOF. Scouting for other
possible metabolites of piracetam, MetabolExpert of the

0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.053
 K. Sahu et al. / European Journal of Medicinal Chemistry 65 (2013) 94e101
Fig. 1. The chemical structure of piracetam (1a), 2-(2-oxopyrrolidin-1-yl) acetic acid
(1b) and the tentative metabolites (1ce1e).
CompuDrug International, Inc. (South San Francisco, USA) was
used. Several metabolites (Fig. 1(b)e(e)) were predicted when
piracetam was subjected to in-silico metabolism [12].
Piracetam can be determined using HPLC [13,14] and LCeMS/MS
[15]. A recent publication [3] gave an account of the determination
of piracetam and 2-(2-oxopyrrolidin-1-yl) acetic acid (M1) in
microsomal media. The analyses were performed using UPLCeMS/
MS on bridge ethylene hybrid (BEH) stationary phase using iso-
cratic elution. The present study contains a UPLCeMS/MS method
for the simultaneous detection of piracetam and M1 in microsomal
preparations and the pharmacokinetics of M1 in vivo. Electrospray
ionization mass spectrometry is preferred to record the ultraviolet
absorbance.
2. Experimental
2.1. Chemicals and solvents
Piracetam was obtained as a gratis sample from Micro Labs Ltd
(Bangalore, India). Metabolite (2-(2-oxopyrrolidin-1-yl) acetic
acid), midazolam and 6-hydroxy midazolam were purchased from
SigmaeAldrich Co. (St Louis, MO, USA). HPLC-grade methanol and
acetonitrile were obtained from the Merck Co. (Darmstadt, Ger-
many). Pooled human liver microsomes from 30 donors were
purchased from BD Gentest (Woburn, MA, USA). Diethyl ether was
purchased from TKM Pharma (Hyderabad, India). Glacial acetic acid
(GAA) AR was purchased from E Merck Limited (Mumbai, India).
TriseHCl, KCl, ammonium acetate, magnesium chloride and ethyl
acetate were all obtained from Merck (Germany). NADPH reduced
tetra sodium salt was purchased from Sisco Research
Laboratories (Mumbai, India). All other chemicals for this study
were of analytical grade and used without further purification.
Prior approval from the Institutional Animal Ethics Committee
(IAEC), Jamia Hamdard (Approval No. 899) was attempted for care
and experimental studies with animals. All experiments, eutha-
nasia, and disposal of carcasses were performed in accordance with
the guidelines laid by IAEC for animal experimentation.
95
2.2. Instrumentation and chromatographic conditions
2.2.1. Ultra performance liquid chromatography (UPLC)
UPLC was performed with a binary solvent delivery pump, an
autosampler and PDA detector of acquity UPLC system manufac-
tured by Waters Corporation (Milford, MA, USA); data were acquired
and processed using Empower software. The chromatographic
separation was performed using a Waters Acquity BEH 10  2.1 mm,
1.7 m column. The column was heated to 30  C and 5 mL of the sample
were injected. Mobile phases were 15% acetonitrileeammonium
acetate buffer (10 mM, pH 5.0). The flow rate was 0.3 ml/min. The
column eluent was monitored by PDA detector at 209 nm. These
parameters were used in the analysis of incubations of piracetam in
the presence of microsomes with NADPH.
2.2.2. Quadrapole-time of flight-mass spectrometry (Q-TOF-MS)
Mass spectrometry was performed on a Waters Q-TOF Premier
(Micromass MS Technologies, Manchester, UK) mass spectrometer.
The nebulization gas was set to 550 L h1, the cone gas was set to
55 L h1 and the source temperature was set to 105  C. The capillary
voltage and sample cone voltage were set to 3.0 KV and 40 V,
respectively. The Q-TOF Premier was operated in V mode with a
resolution over 8600 mass with 1.0 min scan time, and 0.02 s inter-
scan delay. The accurate mass and composition for the precursor
ions and the fragment ions were calculated using the MassLynx V 4.1
software incorporated in the instrument. Argon was employed as the
collision gas at a pressure of 5.3  105 torr. Quantitation was carried
out using Synapt mass spectrometer (Q-TOF) of the transitions of
piracetam and its metabolite. Compounds parameters viz., declus-
tering potential (DP), collision energy (CE), entrance potential (EP)
and collision exit potential (CXP) were 70, 30, 7, 10 V
and 60, 50, 8, 10 V for piracetam and its metabolite, resp
ectively.
2.3. Working solutions and samples preparation
2.3.1. Microsomal studies
The stock solution of 1 mg/ml concentration for piracetam
metabolite (M1) was prepared by dissolving 1 mg of the compound,
made up to a final volume of 1 ml in water. Working stock solutions
were obtained by step-wise dilution of the stock solution in water
to generate working stock solution of 5, 2, 1, 0.75, 0.5, 0.2, 0.1 mg/ml,
and on the day of analysis this set of stocks was used to prepare
standards for the calibration curve. Calibration standards were
prepared by spiking 90 mL of pooled human liver microsomes with
the appropriate working solution of piracetam (10 mL) on the day of
analysis.
2.3.2. Pharmacokinetics studies for metabolite (M1)
Primary stock solutions of 2-(2-oxopyrrolidin-1-yl) acetic acid
(M1) (1 mg/ml) were prepared in water. Working standard solu-
tions of M1 were developed by combining the aliquots of each
primary stock solution and diluting with water. Calibration stan-
dards of M1 (5, 10, 25, 50, 75, 100 and 200 ng/ml) were prepared by
spiking the working standard solutions into pooled drug-free rat
plasma. All the stock solutions were stored at 4  C until analysis.
Quality control (QC) samples were prepared by individually spiking
control rat plasma at three concentration levels [20 ng/ml (QC low),
100 ng/ml (QC medium) and 180 ng/ml (QC high)] and stored
at 75  05  C until analysis.
2.3.3. Sample preparation for pharmacokinetic studies
A single liquideliquid extraction method was followed for
extraction of piracetam from rat plasma. To 100 mL of plasma in a
tube, next a 3 ml aliquot of extraction solvent, ethyl acetate was
96 K. Sahu et al. / European Journal of Medicinal Chemistry 65 (2013) 94e101
added. The mixture was then vortexed for 5 min, accompanied by
centrifugation for 5 min at 2000 g at 20  C on Sigma 3e16 K
(Frankfurt, Germany). The organic layer (2 ml) was separated and
evaporated to dryness under vacuum in speedvac concentrator
(Savant Instrument, Farmingdale, New York, USA). The residue was
reconstituted in 100 mL of the mobile phase and 5 mL was injected
into the UPLCeMS/MS system for analysis.
2.4. In vitro study
The metabolite formation of piracetam was performed using
human liver microsomes. The triplicate glass tubes contain reaction
volume of 500 mL. Incubation of piracetam with human liver mi-
crosomes was carried out at 37  C in a bench-top Lab-Line shaker.
The incubation solution contained 50 mM TriseHCl buffer (pH 7.4),
0.5 mg protein/ml microsomes, 5 mM MgCl2, 2 mM NADPH and
50 mM piracetam in a final volume of 500 mL. The enzyme reaction
was initiated by adding NADPH after an initial 10-min pre-
incubation. The reaction was terminated by adding 2 ml of cold
ethyl acetate at 10, 20, 30, 40 and 50 min. The solution was vortex-
mixed and centrifuged at 4  C for 10 min at 3500 rpm. The super-
natant was transferred to a test tube and evaporated to dryness
under vacuum in the speedvac concentrator. The residue was
reconstituted in 100 mL of the mobile phase, and 5 mL of this solu-
tion was injected to UPLCeMS/MS. The controls groups are vehicle
control (Microsomes incubated without the piracetam but with
vehicle); metabolic positive control (midazolam metabolism to 6-
hydroxy midazolam effective in the presence of cyp3A4) & nega-
tive control (Microsomes combined with the piracetam, and then
ethyl acetate had added before the NADPH).
2.5. In vivo study
The M1 pharmacokinetic study was carried out on Wistar rats.
Piracetam was administered orally at a dose of 50 mg/kg. Blood
samples (Approx. 150 mL) from the retro-orbital plexus were
collected into heparinised microfuge tubes at 0.5, 1, 1.5, 2, 4, 8, 12,
and 24 h post-dosing and plasma was harvested by centrifuging the
blood at 14 000 rpm for 10 min and stored at 20  C until analysis.
In vivo, oral pharmacokinetic study was performed in Wistar
rats (n ¼ 5, weight range 210e230 g) to demonstrate the applica-
bility of newly developed and validated bioanalytical method using
UPLC. Piracetam was administered orally at a dose of 50 mg/kg in
0.25% sodium carboxy methyl cellulose (CMC) suspension. Blood
samples were collected from the retro-orbital plexus of rats under
light ether anesthesia into microfuge tubes containing heparin as
an anti-coagulant at 0.5, 1, 2, 3, 4, 5, 8, 10, 12 and 24 h post-dosing.
Plasma was harvested by centrifuging the blood at 2500 g for 5 min
at 20  C and stored at 75  10  C until analysis. Rat plasma (100 mL)
samples were processed as described above and data was accepted
based on the performance of QCs prepared to use rat blank plasma
(three QCs each at three concentration levels). The criteria for
acceptance of the analytical runs encompassed the following: (1)
not more than 33% of the QC samples were greater than 15% of the
nominal concentration; (2) not less than 50% at each QC concen-
tration level must meet the acceptance criteria. Plasma concen-
trationetime data of piracetam metabolite M1 were analyzed by
the non-compartmental method using WinNonlin Version 5.1
(Pharsight Corporation, Mountain View, California, USA).
2.6. Validation procedures in plasma
A strong validation according to the FDA protocols was carried
out for the assay in rat plasma [3,16,17].
2.6.1. Specificity and selectivity
The specificity of the method was evaluated by analyzing rat
plasma samples collected from six rats to investigate the potential
interferences at the UPLC peak region for analyte using the pro-
posed extraction procedure for chromatographic conditions [17].
2.6.2. Matrix effect
The effect of rat plasma constituents over the separation of
piracetam and its metabolite being determined by comparing the
responses of the post extracted plasma standard QC samples (n ¼ 6)
with the response of analytes from neat standard samples at cor-
responding concentrations. The matrix effect for piracetam was
determined at QC low, QC medium, and QC high concentrations,
viz., 20, 100 and 180 g/ml.
2.6.3. Calibration curve
The calibration curve was acquired by plotting the ratio of the
peak area versus the nominal concentration of calibration stan-
dards. The final concentrations of calibration standards obtained
for plotting the calibration curve were 10, 20, 50, 75, 100, 200 ng/
ml. The results were fitted to linear regression analysis using 1/X2 as
a weighting factor. The calibration curve had to have a correlation
coefficient (r) of 0.995 or better. The acceptance criteria for back-
calculated standard concentration were 15% deviation from the
nominal value except at LLOQ, which was set at 20% [16,17].
2.6.4. Precision and accuracy
The intra-day assay precision and accuracy were estimated by
analyzing six replicates at four different QC levels, i.e., 10, 100 and
180 ng/ml. The inter-day assay precision was determined by
analyzing the four levels QC samples on three different runs. The
criteria for acceptability of the data included accuracy within 15%
standard deviation (S.D.) from the nominal values and precision of
within 15% relative standard deviation (R.S.D.), except for LLOQ,
where it should not exceed 20% of accuracy as well as precision.
2.6.5. Stability experiments
All stability studies were conducted at two concentration levels,
i.e. QC low and QC high, using six replicates at each concentration
levels. Replicate injections of processed samples were analyzed up
to 18 h to establish auto-sampler stability of analyte at 4  C. The
peak area of analyte obtained at initial cycle was used for the
reference to determine the stability at subsequent points. The sta-
bility of piracetam in the biomatrix during 4 h exposure at room
temperature in rat plasma (bench top) was determined at ambient
temperature (25  2  C). Freeze/thaw stability was evaluated up to
three cycles. In each cycle, samples were frozen for at least 12 h
at 075  10  C. Freezer stability of piracetam in rat plasma was
assessed by analyzing the QC samples stored at 75  10  C for at
least 15 days. Samples were considered to be stable if assay values
were within the acceptable limits of accuracy (i.e., 15% SD) and
precision (i.e. 15% RSD).
2.6.6. Recovery
The extraction recovery of analytes, through liquideliquid
extraction procedure, was determined by equating the peak areas
of extracted plasma (pre-spiked) standard QC samples (n ¼ 6) to
those of the post-spiked standards at equivalent concentrations.
Recoveries of M1 were determined at three concentration levels QC
low, QC medium and QC high concentrations viz., 20, 100, and
180 ng/ml.
2.6.7. Statistical analysis
Experimental values are expressed as mean  SD. The phar-
macokinetic parameters were determined non-compartmental
 K. Sahu et al. / European Journal of Medicinal Chemistry 65 (2013) 94e101
Table 1
Retention times and relative retention times of various peaks in UPLC.
Metabolite RT RRT Observed molecular MS/MS
weight pattern
M1 2.042 0.749 143.14 144.08, 127.08
Piracetam 2.726 1.000 142.16 143.10, 126.07
UPLC ¼ ultra performance liquid chromatography.
method using WinNonlin Version 5.1 (Pharsight Corporation,
Mountain View, California, USA).
3. Results and discussion
3.1. Development and optimization of the UPLC method
Initially, the drug was analyzed on a BEH C18 column using
acetonitrile: water (50:50) as a mobile phase at a flow rate of
0.3 ml/min and a column temperature of 25  C. Under these con-
ditions, the shape of this drug peak was not acceptable. Subsequent
trials were made on microsomal samples using different amounts
of acetonitrile, pH, and temperature. The best separation was ach-
ieved on the same column at 30  C. Mobile phase consisting of
acetonitrile and ammonium acetate buffer (10 mM, pH 5) in the
ratio of 15:85 (v/v) at a flow rate of 0.3 ml/min was found to be
appropriate during LC optimization and enabled the determination
of the electrospray response for piracetam and its metabolite. The
detection wavelength was 209 nm at constant injection volume of
5 mL for UPLC. The retention times (RT) and relative retention times
(RRT) for piracetam and its metabolite are shown in Table 1. The
standard chromatogram of UPLC contains the peak of piracetam
and its metabolite obtained from the analysis of microsomal sam-
ples is shown in Fig. 2.
3.2. Mass spectrometry
To optimize ESI conditions for piracetam and its metabolite, Q-
TOF full scans were carried out in positive ion detection mode. The
sample introduction (infusion mode) experiment, the mass spectra
for piracetam and its metabolite revealed peaks at m/z 143 amu and
144 amu respectively as protonated molecular ions [M þ H]þ. The
product ion mass spectrum for piracetam and its metabolite (M1)
are shown in Fig. 3(a) and (b) respectively. With the help of UPLC
Fig. 2. Chromatogram shows separation of piracetam and its metabolite in a mixture of microsomal preparations.
97
coupled with MS analysis, a small amount of polar M1 at RT
2.042 min was detected. The M1 responded better to positive ESI
mode, and the full-scan mass spectrum presented [M þ H]þ at m/z
144.08 amu as the main ion and a major fragment ions at m/z
127.08 amu. Therefore, the m/z 144.08 / 127.08 transition was
selected for M1 and the m/z 143.10 / 126.07 for piracetam for
further LCeMS/MS analysis in multiple reactions monitoring
(MRM) mode in the MassLynx software.
3.3. In silico studies
A well established practice of prediction of drug metabolism is
the use of in silico methods: the computer aided prediction of
metabolism. The presumed MetabolExpert program screens for
potential metabolites on the basis of known metabolic alterations
as well as steric possibilities. One of the major advantages of in silico
metabolism studies is to provide prediction of metabolites that are
likely to be formed in vivo and in vitro. Recent trends in metabolic
research appear to favor the use of in vitro microsomal metabolism
studies. The liver microsomal preparations are derived from cell
cultures and allow the high throughput metabolite recognition.
While microsomal metabolism methods, usually do not provide a
complete picture of in vivo metabolic alterations valuable infor-
mation concerning metabolic stability and essential metabolites of
the drug. Especially, the oxidative and the hydrolytic metabolites
appear in the sample derived from microsomal preparation. Several
metabolites (Fig. 1(b)e(e)) were predicted when the piracetam was
subjected to in silico studies. However only the existence of M1
(Fig. 1(b)) was proven in in vivo and in vitro studies.
3.4. Studies of NADPH-dependent metabolism in human liver
microsomes
Metabolism of piracetam was investigated in incubations of
50 mM in human liver Microsomes at different time 10, 20, 30, 40
and 50 min. The experimental findings indicate that a human liver
microsomal preparation (in the presence of NADPH) metabolizes an
essential portion of the parent compound. With the help of MS
analysis, small, more polar, metabolite at RRT 0.749 min was
detected. The detectable metabolite is 2-(2-oxopyrrolidin-1-yl)
acetic acid, the compound formed by the hydrolysis of the ester of
the parent piracetam. The UPLC chromatogram in Fig. 2 also shows
additional a peak of the metabolite at RT 2.042 (RRT 0.749) along
98 K. Sahu et al. / European Journal of Medicinal Chemistry 65 (2013) 94e101

Fig. 3. MS/MS product-ion spectrum of piracetam and its metabolite with [M þ H]þ at m/z 143.10 and 144.08 as the precursor ion, respectively.

with a peak of the parent compound at 2.726 (RRT 1.000) that
confirms the formation of the metabolite. Further it was confirmed
by using MS/MS analysis. The peaks at RT 2.042 and RT 2.726 were
checked in MS analysis for the molecular weight of metabolite (M1)
and piracetam respectively. On the basis of obtained molecular
weight, the mechanism of formation of metabolite was proposed.
The proposed metabolite was further confirmed by using standard
metabolite. The common application of isocratic elution and mul-
tiple reaction monitoring methods made possible to the differen-
tiate piracetam and its metabolite. Table 2 gives data for the in vitro
metabolism of piracetam using human liver microsomal prepara-
tion at different time interval of 50 mM concentration. The
 K. Sahu et al. / European Journal of Medicinal Chemistry 65 (2013) 94e101 99

Table 2 3.5. Validation procedures

Data depicts the formation of metabolite at different time interval in human liver
microsomes.
3.5.1. Calibration curve
Time (min.) Piracetam mM (mg/ml) M1 mM (mg/ml) The linear regressions of the peak area versus concentrations
0 50.00 (7.108) 00.00 (0.00) were fitted over the concentration range 10e200 ng/ml for M1 in
10 47.56 (6.761) 02.42 (0.346) rat plasma. A typical equation of the calibration curve was:
20 45.66 (6.491) 04.79 (0.686) y ¼ 1.694x þ 2.051, r2 ¼ 0.9981, where y represents M1 peak area
30 43.78 (6.224) 06.45 (0.923)
and x represents the plasma concentration using weighing factor
40 40.56 (5.766) 09.23 (1.321)
50 36.22 (5.149) 13.89 (1.988) (1/X2).

3.5.2. Specificity, recovery and matrix effect

The specificity and selectivity have been studied by using in-
dependent plasma samples from six different rats. As shown in
Fig. 2, there is no significant interference from plasma found at
retention time of either the analyte or its metabolite. The RT and RRT
of piracetam and its metabolite are shown in Table 1.
The extraction recovery was determined by comparing the peak
areas of pre-spiked standards at 20, 100 and 180 ng/ml with those
of post-extraction blank plasma standards spiked with corre-
sponding concentrations. Mean recoveries of M1 were 92.2, 96.3
and 97.6% (n ¼ 5) at concentrations of 20, 100 and 180 ng/ml,
respectively.
Fig. 4. Proposed metabolic pathway of piracetam in human liver microsomes.
The matrix effect was evaluated by analyzing QC low (20 ng/ml),
QC medium (100 ng/ml) and QC high samples (180 ng/ml). Average
matrix effect values were 94.25%, 97.49% and 98.18%.
Table 3
Precision and accuracy for M1 of quality control sample in rat plasma (N ¼ 5). 3.5.3. Precision and accuracy
Nominal concentration Observed concentration Precision (%)a Accuracy(%)b The precision of the method was determined by calculating RSD
(ng/ml)
Mean SD
for QCs at three concentration levels over two validation days. The
inter-day and intra-day precision was found to be a range of 0.054e
Inter-day
2.533 and 0.422e4.762 respectively at all QC level (20, 100, and
20 19.860 0.503 2.533 99.300
100 98.967 0.053 0.054 98.967 180 ng/ml). The accuracy of the method ranged from 97.217 to
180 179.603 1.217 0.678 99.780 99.780% at each QC level. Assay performance data are presented in
Intra-day Table 3. The above results demonstrate that the values are within
20 19.463 0.927 4.762 97.317 the acceptable range, and the method is accurate and precise.
100 98.250 0.526 0.535 98.250
180 178.083 0.751 0.422 98.935
a
3.5.4. Stability
Expressed as % R.S.D. ¼ (S.D./mean)  100.
b The stability results showed that piracetam spiked into rat
Calculated as (mean determined concentration/nominal concentration)  100.
plasma was stable for 2 h at room temperature, for 30 days
at 20  C, and during three freezeethaw cycles. Stability of pira-
mechanism of formation of the metabolite is shown in Fig. 4. The cetam extracts in the sample solvent on an autosampler was also
fast isocratic elution UPLC with MS-MRM monitoring allows observed over a 24 h period. The results of stability experiments are
detecting the piracetam and M1 in samples metabolized using listed in Table 4.
human liver microsomal preparations. The experimental findings
indicate that a human liver microsomal preparation metabolizes an
amide portion of piracetam to carboxylic acid in higher concen- 3.6. Pharmacokinetic
tration at 50 min. The detectable metabolite (M1) is 2-(2-
oxopyrrolidin-1-yl) acetic acid, the M1 formed by the hydrolysis The developed LCeMS/MS method is used to determine the M1
of the amide group of the piracetam. concentrations in plasma samples from Wistar rats after an oral

Table 4
Summary of stability of M1 under various storage conditions (N ¼ 5).

Condition Spiked concentration Found concentration Precision Accuracy (%)b RE (%)c

(ng/ml) (ng/ml) (%)a

Ambient, 2 h 20 19.347 1.520 96.733 3.267

180 178.883 0.437 99.380 0.620
20  C, 30 days 20 18.787 1.298 93.933 6.067
180 179.123 0.107 99.513 0.487
Three freezeethaw 20 18.880 0.377 94.400 5.600
180 178.420 0.269 99.122 0.878
Autosampler ambient 24 h 20 19.043 2.187 95.217 4.783
180 178.753 0.133 99.307 0.693
10 days, 80  C 20 18.853 1.804 94.267 5.733
180 179.130 0.170 99.517 0.483
a
Expressed as % R.S.D. ¼ (S.D./mean)  100.
b
Calculated as (mean determined concentration/nominal concentration)  100.
c
Calculated as (observed concentration  nominal concentration/nominal concentration)  100.
100 K. Sahu et al. / European Journal of Medicinal Chemistry 65 (2013) 94e101
Fig. 5. Mean plasma concentration time profile after oral administration of piracetam
metabolite.
Table 5
Pharmacokinetic parameters of M1 after oral administration of
single dosage 50 mg/kg piracetam to rats (n ¼ 5).
Parameters Observations
Cmax (ng/ml) 155.390
Tmax (h) 001.000
AUC0et (mg h/ml) 928.445
AUC0eN (mg h/ml) 939.178
CL (L/h/kg) 053.238
AUC%Extrap 001.141
t1/2 (h) 003.652
MRTt 005.542
administration of piracetam. The isocratic elution UPLC with MS-
MRM monitoring provides reliable detection of M1 in rat plasma
samples. The typical representative spectrum of treated rat plasma
was shown in Fig. 3. The m/z 143.10 amu precursor ion to the m/z
126.07 amu was used for quantification for piracetam. Similarly, for
M1 m/z 144.08 amu precursor ion to the m/z 127.44 amu was used
for the quantification purpose. The mean plasma concentratione
time curve after administration of a single 50 mg/kg oral dose of
piracetam for its metabolite (M1) is shown in Fig. 5. The main
pharmacokinetic parameters from non-compartment model anal-
ysis are summarized in Table 5. Plasma data were subjected to non-
compartmental pharmacokinetics analysis using WinNonlin
(version 5.1, Pharsight Corporation, Mountain View, USA). The
observed maximum plasma concentration (Cmax ¼ 155.390 ng/ml)
and the time to reach the maximum plasma concentration
(Tmax ¼ 1.000 h) were obtained by visual inspection of the exper-
imental data. The area under the plasma concentration time curve
(AUC0et ¼ 928.445 ng h/ml) was calculated using a linear trape-
zoidal method. The apparent elimination half-life (t1/2 ¼ 3.652 h)
was calculated as 0.693/kel and the kel was estimated by linear
regression of the log of plasma concentrations in the terminal
phase. The clearance (CL) of M1 was 53.238 L/h/kg, while the
minimum residual time (MRTt) was 5.542 h.
4. Conclusion
An efficient bioanalytical method based on fast isocratic UPLCe
MS/MS method has been demonstrated for the simultaneous
determination of piracetam and its phase I metabolite i.e., 2-(2-
oxopyrrolidin-1-yl) acetic acid (M1) in human hepatic micro-
somal preparations. Additionally, development of a validated
method for M1 in rat plasma samples by UPLCeMS/MS and applied
on pharmacokinetic analysis. The method was successfully applied
to a pharmacokinetic study of the piracetam metabolite after oral
administration of piracetam (50 mg/kg). The formed metabolite of
piracetam was quantified that are generated after the enzymatic
hydrolysis. The predicted metabolite from in silico studies also
suggests that the formed metabolite is 2-(2-oxopyrrolidin-1-yl)
acetic acid (M1). Based on these results, 2-(2-oxopyrrolidin-1-yl)
acetic acid is the sole metabolite of piracetam in Phase I metabolism
has been confirmed. The plasma concentration versus time profile
of metabolite of piracetam in rats has been determined following
orally administration of piracetam, and it has been shown that
piracetam metabolizes to 2-(2-oxopyrrolidin-1-yl) acetic acid
in vivo. The developed bioanalytical method has extended appli-
cability viz., various in vivo and in vitro metabolism (Phase I and II)
and would be suitable for clinical medical study.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgments
The authors are grateful to the Vice Chancellor, Jamia Hamdard,
New Delhi for providing the experimental facilities for this research
study. Mr. Kapendra Sahu wishes to thank the Department of Sci-
ence & Technology (DST), New Delhi, for providing him research
fellowship.
References
[1] G. Caldwell, Z. Yan, Optimization in Drug Discovery: In Vitro Methods,
Humana Press, Totowa, N.J., 2004.
[2] Y. Kwon, Handbook of Essential Pharmacokinetics, Pharmacodynamics, and
Drug Metabolism for Industrial Scientists, Kluwer Academic/Plenum Pub-
lishers, New York, 2001.
[3] K. Sahu, M. Shaharyar, A.A. Siddiqui, Effect of morin on pharmacokinetics of
piracetam in rats, in vitro enzyme kinetics and metabolic stability assay using
rapid UPLC method, Drug Test. Anal. (2012).
[4] M.J. O’Neil, The Merck Index: An Encyclopedia of Chemicals, Drugs, and Bi-
ologicals, thirteenth ed., Merck, Whitehouse Station, N.J., 2001.
[5] M.Z. Wang, J.Y. Saulter, E. Usuki, Y.L. Cheung, M. Hall, A.S. Bridges, G. Loewen,
O.T. Parkinson, C.E. Stephens, J.L. Allen, D.C. Zeldin, D.W. Boykin, R.R. Tidwell,
A. Parkinson, M.F. Paine, J.E. Hall, CYP4F enzymes are the major enzymes in
human liver microsomes that catalyze the O-demethylation of the antipara-
sitic prodrug DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidox-
ime], Drug Metab. Dispos. 34 (2006) 1985e1994.
[6] B. Dalmadi, J. Leibinger, S. Szeberenyi, T. Borbas, S. Farkas, Z. Szombathelyi,
K. Tihanyi, Identification of metabolic pathways involved in the biotransfor-
mation of tolperisone by human microsomal enzymes, Drug Metab. Dispos. 31
(2003) 631e636.
[7] W.Z. Shou, L. Magis, A.C. Li, W. Naidong, M.S. Bryant, A novel approach to
perform metabolite screening during the quantitative LCeMS/MS analyses of
in vitro metabolic stability samples using a hybrid tripleequadrupole linear
ion trap mass spectrometer, J. Mass. Spectrom. 40 (2005) 1347e1356.
[8] R. Dams, M.A. Huestis, W.E. Lambert, C.M. Murphy, Matrix effect in bio-
analysis of illicit drugs with LCeMS/MS: influence of ionization type, sample
preparation, and biofluid, J. Am. Soc. Mass. Spectrom. 14 (2003) 1290e1294.
[9] Wahajuddin, S.P. Singh, G.K. Jain, Determination of lumefantrine in rat plasma
by liquideliquid extraction using LCeMS/MS with electrospray ionization:
assay development, validation and application to a pharmacokinetic study,
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877 (2009) 1133e1139.
[10] A. Tolonen, M. Turpeinen, O. Pelkonen, Liquid chromatographyemass spec-
trometry in in vitro drug metabolite screening, Drug Discov. Today 14 (2009)
120e133.
[11] D. Zhang, M. Zhu, W.G. Humphreys, Drug Metabolism in Drug Design and
Development, John Wiley & Sons, Inc., Chichester, 2007, p. 1. online resource
(633 pp.).
[12] H. Kalasz, G. Petroianu, K. Tekes, I. Klebovich, K. Ludanyi, Z. Gulyas, Meta-
bolism of moexipril to moexiprilat: determination of in vitro metabolism
using HPLC-ES-MS, Med. Chem. 3 (2007) 101e106.
[13] M.S. Arayne, N. Sultana, F.A. Siddiqui, A.Z. Mirza, F. Qureshi, M.H. Zuberi,
Simultaneous determination of piracetam and its four impurities by RP-HPLC
with UV detection, J. Chromatogr. Sci. 48 (2010) 589e594.
[14] A. Curticapean, S. Imre, New validated method for piracetam HPLC determi-
nation in human plasma, J. Biochem. Biophys. Methods 69 (2007) 273e281.
[15] X. Wang, J. Zhu, R. Xu, X. Yang, H. Wu, D. Lin, F. Ye, L. Hu, Determination of
piracetam in rat plasma by LCeMS/MS and its application to pharmacoki-
netics, Biomed. Chromatogr. 24 (2010) 1108e1112.
 K. Sahu et al. / European Journal of Medicinal Chemistry 65 (2013) 94e101 101

[16] S.P. Singh, Wahajuddin, D.K. Yadav, P. Rawat, R. Maurya, G.K. Jain, Quantitative [17] Center for Drug Evaluation and Research (U.S.), Center for Veterinary Medi-
determination of formononetin and its metabolite in rat plasma after intrave- cine (U.S.), Guidance for Industry Bioanalytical Method Validation, U.S. Dept.
nous bolus administration by HPLC coupled with tandem mass spectrometry, of Health and Human Services, Food and Drug Administration Center for
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 878 (2010) 391e397. Veterinary Medicine, Rockville, MD, 2001, p. 1. online resource (22 pp.).