Academic Sciences
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491
Vol 4, Suppl 5, 2012
Research Article
BIO-ANALYTICAL METHOD DEVELOPMENT AND ITS VALIDATION FOR ESTIMATION OF
PHENOBARBITAL IN HUMAN PLASMA USING LIQUID CHROMATOGRAPHY COUPLED WITH
TANDEM MASS
SMRITI PANDEYb, AJIT KUMAR YADAV*a, SUNIL SINGHa, HEMENDRA GAUTAMa AND SURABHI SHARMAa
aDepartment
of Pharmacy, Invertis Institute of Pharmacy, Invertis University, Bareilly-243 123, Uttar Pradesh, India, bDepartment of
Pharmaceutical Chemistry, NRI Institute of Pharmacy, Raisen Road, Bhopal-462021, Madhya Pradesh, India.
Email: ajit.y@invertis.org , rssunil29@rediffmail.com
Received: 26 July 2012, Revised and Accepted: 08 Sep 2012
ABSTRACT
A high throughput and sensitive liquid chromatographytandem mass spectrometry (LCMS/MS) method has been developed and validated for the
estimation of Phenobarbital in human plasma. Phenobarbital was extracted from human plasma using solid-phase extraction technique using
Phenytoin Sodium as internal standard. A Beta basic 8 column provided chromatographic separation of analyses followed by detection with mass
spectrometry. The mass transition ion-pair was followed as m/z 251.4 98.0for Phenobarbital. The method involves a simple multiplexing, rapid
solid-phase extraction, simple isocratic chromatography conditions and mass spectrometric detection which enable detection at sub-nanogram
levels. The proposed method has been validated for a linear range of 5.5 10.3 ng/mL with correlation coefficient0.99 79. The precision and
accuracy were within 10% for intra-HPLC runs and inter-HPLC runs. The overall recoveries for Phenobarbital were 99.89%. Total MS run time was
4 min. The developed method was applied for the determination of pharmacokinetic parameters of Phenobarbital following a single oral
administration of a 30 mg Phenobarbital tablet in human plasma.
Keywords: LC/MS/MS, Phenobarbital, HPLC, Validation, Human Plasma.
INTRODUCTION
MATERIALS AND METHODS
Phenobarbital (PBT) chemically 5-ethyl-5-phenylpyrimidine2,4,6(1H,3H,5H)-trione1 belongs to anticonvulsant drugs it also has
sedative and hypnotic properties2 (Figure. 1) but, as with other
barbiturates, has been superseded by the benzodiazepines for these
indications3. Phenytoin Sodium an antiepileptic drug was used as an
internal standard. The mass transition ion-pair for Phenobarbital
(PBT) m/z 251.4 98.0. Different methods have been reported in
the literature for monitoring plasma levels of PBT4, and in dosage
forms also5. Some other techniques used in individual analysis of
PBT from plasma include HPLC with Mass Spectroscopy, UVSpectroscopy 6,7,8,9, but these are not sensitive. Since there is no
specific method was available in literature for the quantification of
Phenobarbitol in human plasma using LC/MS/MS system, the study
was done by different literature survey9,10,11,12,13,14,15 the aim of the
study was the development and validation of simple, sensitive, rapid
and specific method.
Chemicals used
Acetonitrile (Merck), Methanol (Merck), Millipore water were used
in method development.
Instrumentation
API 3000 LC/MS/MS was used.
Mass Parameters
The mass spectrometer was operated in the positive ion mode. The
developed mass and LC parameters for the estimation of PBT and
Phenytoin Sodium (PTS) as internal standard are given below. The
compound dependent parameters for Phenobarbitol was 55eV,
Focussing potential was 160eV, Entrance potential 10eV, Collision
energy CE 30 psi. For Phenytoin Sodium potential was 50eV,
Focusing potential was 190 eV, Entrance potential 10 eV, Collision
energy CE 100 V. Source dependent parameters for the method are
CUR -15 psi, TEMP -550c, ISV-5500 V, CAD-18psi.
Multi Reaction Monitoring (MRM)
Fig. 1: Chemical structure of Phenobarbital
(a)
The mass transition ion-pair has been followed as m/z 251.4 98.0
for PBT and m/z 423.291.0 for PTS.
(b)
Fig. 2: Mass spectrum of Phenobarbital (a) Parent ion (b) Daughter ion
Yadav et al.
(a)
Int J Pharm Pharm Sci, Vol 4, Suppl 5, 288-292
(b)
Fig. 3: Mass spectrum of Phenytoin sod. (a) Parent ion (b) Daughter ion
From the chromatogram above the retention time (Rt) for Phenytoin Sodium was found to be 0.87 min and Phenobarbitol 0.69 min.
LC parameters
Column: Hypurity advanced 504.6 mm, 5 m (thermo)
Mobile phase: Acetonitrile:methanol (50:50)
Run time: 4.0 min.
Injection volume: 5 L
Flow Rate: 600 L /min.
Column oven temperature: 40c
Time
Flow
0
2.5
3
4
600l/min
600l/min
600l/min
600l/min
Table 1: Gradient System
Mobile Phase
A%
95
95
30
30
Mobile Phase
B%
5
5
70
70
Fig. 4: Chromatogram for Optimization of Mobile Phase (Water: Acetonitrile) [(a) Phenobarbital, (b) Phenytoin Sodium (ISTD).
Preparation of calibration curve standards and quality controls
for estimation of Phenobarbitol
To 96l of plasma 4L of standard solution (SS) of Phenobarbitol
was added and vortexed. The spiked plasma samples were pooled
and 100L was taken from it. To it acetonitrile containing IS
(Phentoin Sodium 200 ng/ml) was added, vortexed and kept for
centrifugation which rotates at 13000 rpm at 5c. The supernatant
was taken and transferred to plates for analysis. In similar way
remaining calibration standards and quality controls were prepared
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Yadav et al.
to get calibration curve standards with arrange of 5.5% to 10.3%
ng/ml.
Method validation
Objective
System suitability
The objective of the work is to validate specific LC/MS/MS method
for the determination of PBT in human plasma for the study. Injector
carries over effect for analyte and is Carry over test was performed
in the following sequence.
Six replicates of Extracted standard 9 (ULOQ) sample was injected
Results are presented in Table 2.
Table 2: System suitability analysis
Replicate
1
2
3
4
5
6
Mean
SD
% CV
Int J Pharm Pharm Sci, Vol 4, Suppl 5, 288-292
MP STD9 MP MPSTD1 Plasma Blank.
Analyte Area/IS area
7.905695
8.096845
7.956168
8.743318
8.384934
8.468316
8.259213
0.327738
3.968151
No significant injector carries over was observed for PBT and
internal standard. Results are presented in table 3.
Blank matrix screening (Specificity)
During validation, blank plasma samples from 4 different lots were
processed according to the extraction procedure and evaluate the
interference at the retention times of analytes and internal standard.
The 3 free interference lots were selected from the 4 lots. Presented
in table 4.
Acceptance Criteria(s)
Extraction recovery
The % CV of the drug/IS area ratio of last five injections should not
be more than 5.0
The percentage recovery of PBT and PTS was determined by
comparing the mean peak area of PBT in extracted LQC, MQC, HQC
samples with freshly prepared un-extracted LQC, MQC, HQC
samples. The percentage recoveries were found to be above 75%.
Conclusion: System is suitable for determination of Phenobarbital
from human blood plasma.
Table 3: Assessment of Injector carry over effect for Analyte PBT and IS (PTS)
Sample
Identification
MP
S9
MP
MP
S1
Plasma Blank
Analyte
Response
0
673905
0
0
15562
0
Internal standard
Response
0
779645
0
0
875943
0
Result: Injector carry over effect for Analyte (PBT) and IS was passed
Carry Over observed
with Analyte
Nil
Nil
Nil
Nil
Nil
Nil
Carry Over observed
with IS
Nil
Nil
Nil
Nil
Nil
Nil
Acceptance criteria
The carry over must be less than 20% response of analyte (S1) and less than 5% response of internal standard.
Table 4: Screening of Different batches of blank matrix (HUMAN PLASMA) Screening (SPECIFICITY)
S. No.
1
2
3
4
5
6
7
8
9
10
Mean
Response of signal
(Height) (S)
1770.000
1755.000
1542.000
1743.000
1698.000
1648.000
1769.000
1779.000
1691.000
1780.000
-
Acceptence criteria: Signal (LLOQ)/ noise ratio NLT 5:1
Analysis of subject samples preparation (Extraction procedure)
A 100L of subject plasma sample was mixed with 300L of internal
standard working solution (200ng/ml of PBT) vortexed for two
minutes and kept in centrifuge for 5 minutes at 5C which rotates at
a speed of 13000 rpm. After that the supernatant layer was collected
and analyzed in LC/MS/MS system. The chromatograms of mobile
phase , Blank plasma, Internal standard, Calibration standards,
subject samples and table representing time points vs. plasma drug
concentrations are shown in illustrations.
RESULTS AND DISCUSSION
The goal of present work was to develop and validate a simple,
rapid, sensitive method for extraction and quantification of PBT, was
Response of noise
(Height) (N)
12.300
15.000
11.000
11.250
13.010
10.500
12.550
10.990
13.840
13.170
-
Signal to
Noise ratio S/N
143.90
117.00
140.18
154.93
130.51
156.95
140.96
161.87
122.18
135.16
140.37
suitable for pharmacokinetic studies. To achieve the goal different
options was evaluated to optimize sample extraction, detection
parameters and chromatography. Different columns (X bridge C18,C8,
Phenomenex C18,C8 but polar RP column was found to be better with
minimum tailing and good retention. Coming to the mobile phase the
combination of methanol and Acetonitrile has good eluting capacity
and is cheaper. When acetonitrile is used the analytes are not at all
retaining. A flow rate of 600 L /min was optimized for good
retention and the column oven temperature was kept at 40c. The
LOD, LOQ values are also low (5.01ng/ml) indicating that method is
very sensitive. The injection carry over, specificity, system suitability
was in limits. While coming to extraction procedure precipitation
was found to be better than Liquid-liquid extraction with good
recoveries.
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Int J Pharm Pharm Sci, Vol 4, Suppl 5, 288-292
Fig. 5: Mobile Phase Chromatogram
Fig. 6: Blank plasma chromatogram of losartan calibration curve
CONCLUSION
A simple, specific, rapid and sensitive analytical method was
developed for the determination of PBT in human plasma. Most of
the analytical methods reported, for quantization of PBT individually
from human plasma, require laborious extraction procedure like
liquidliquid extraction, long run time and high quantification limit.
The method was provided excellent specificity and linearity. The
other major advantage of this method is the short run time of 4 min
which allows the quantization of over 300 plasma samples per day.
The recoveries in the method were also good.
ACKNOWLEDGEMENT
This study was supported by the Auriga Research, Kirti Nagar,
New Delhi. and also thankful to NRI Institute of Pharmacy, Bhopal
which provide platform for work.
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