Development and validation of Bioanalytical Method for Quantification of phenytoin in rat brain tissues as per ICH guidelines
CH. Naveen Kumar1*, N.
Kannappan2
1Teegala
Krishna Reddy College of Pharmacy, Department of Pharmaceutical Analysis &
Quality Assurance, Medbowli, Meerpet,
Saroornagar, Hyderabad, Telangana, India.
2Department
of Pharmacy, Faculty of Engineering and Technology, Annamalainagar,
Annamalai University, Chidambaram, Tamilnadu, India.
*Corresponding Author E-mail: naveen2626@gmail.com
ABSTRACT:
Phenytoin is an antiepileptic drug
approved in the USA, Europe and several other countries. Phenytoin
is currently used to manage partial onset seizures in humans suffering from
epilepsy. A rapid, sensitive, novel and simple UV method was developed and
validated according to ICH guidelines for the quantification of phenytoin in rat brain homogenate supernatant. Calibration
curves were found to be linear ranging from0.02-0.1 µg/ml giving regression of
r2=0.998, using detection wavelength at 214 nm. Precision, limit of detection
and limit of quantitation were calculated. Recovery
ranged from 96.38% to 98.55% w/w. The assay was applied successfully to a pre-clinical
study of Phenytoin. By applying this method, we were
able to determine the brain concentration of phenytoin
during at least 1 h after IV administration of 1mg/kg phenytoin.
The method proved to be simple, useful and appropriate, for preclinical and
experimental research for various biomedical applications.
KEYWORDS: Phenytoin, brain
tissue, bioanalytical method validation,RP-HPLC,
Quantification.
1. INTRODUCTION:
A bioanalytical
method offers a consolidated study of, bioequivalence and bioavailability
pharmacokinetic, toxicokinetics studies at various
stages of drug discovery program and support pre clinical and phases I to phase
IV clinical trials [1] is a set of procedures
involved in the study collection,
processing, storage, and analysis of a
biological matrix for a chemical compound study. Bioanalytical
method validation (BMV) is the process used to establish that a quantitative
analytical method is suitable for biomedical analysis applications.
Reassurances as to the quality of the optimized method and its reliability come
from adopting a minimum series of validation experiments and
obtaining satisfactory results.
Characterizing the stability of analytes in
biological samples collected during clinical studies together with that of critical
assay reagents, including analyte stock solutions, is
recognized as an important component of bioanalytical
assay method validation.
Bioanalytical method validation includes all of the
procedures that demonstrate that a particular method used for quantitative
measurement of analyte in a given biological matrix,
such as blood, plasma, serum, or urine, is reliable and reproducible for the
intended use. Phenytoin has a maximum bioavailability
with minimal first-pass metabolism and serum protein binding. Approximately 40%
of the drug is ultimately excreted by the kidney with the remainder cleared by
metabolism. Phenytoin is functionalized amino acid
that acts by selectively enhances slow inactivation of voltage-gated sodium
channels and interacts with collapsing response mediator protein-2, a protein
mainly expressed in the central nervous system and involved in neuronal
differentiation and axonal outgrowth. [2,3] there are very few pharmacokinetic data,
especially on central nervous system [4-7] however, none of these methods
included the analysis of Phenytoin in rat brain
tissue. The aim of this paper was to establish a simple and sensitive UV
procedure for determination of Phenytoin in rat brain
tissue.
2. EXPERIMENTAL:
2.1. Apparatus
A UV
– Visible double beam spectrophotometer (Analytical Technolgies)
K-20155M Model with 10 mm quartz cell were used for experiment and the
calibration for instrument is done prio to the
experiment as per standard calibration procedure.
2.2. Reagents and materials:
Phenytoin.All the reagents and chemicals used were of analytical grade.
2.3. Animals
and drug treatments
The
ultraviolet spectrophotmetric analytical method was developed for quantification of phenytoin from
albino rat brain after oral administration
of phenytoin to
male swiss
Albino rats (9 weeks of age, body weight
that were obtained from Breeder. The proposed experimental studies were
performed according to the guidelines of Institutional Ethics Committee (IEC)
and followed CPCSEA under Registered permission number for this present work is
202/ 2014/GNIP/CPCSEA. Animals were
kept in cages with free access to standard mice diet and water. The animals
were maintained at a temperature of 25–30°C with a 12 h light/dark cycles.
Before the test, animals were fasted prior to dosing by withholding food
overnight, but not water. Phenytoin was dissolved in
vehicle DMSO was given as a single oral dose (1mg/kg) to mices.
The Animals were not subjected to any pharmacological treatment were used as a
source of drug-free rat brain tissues,
which were used as blank matrices in the analytical validation studies. [8]
2.3.1. Sample preparation
A
procedure for the isolation of Phenytoin from brain
samples prior to UV method development. Mice brain collection was done after
1hr of dosing. Each mice was anaesthetized with ether and sacrificed,
decapitated and the whole brain was eased out of the skull. Drug-free (i.e.
blank) brain tissue was also
obtained from the
control rats which were
injected with solvent
alone. Drug-free subsequently added to each brain homogenate and the
organic layer centrifugation and the supernatants was directly used for
analysis [9] After sacrifice removal of
brain was done , it was rinsed with cold saline(0.5 % NaCl,
g/ml) then surface vasculature was ruptured then blotted with dry gauze and weight was taken
and recorded . The whole brain is homogenized within 1hour of collection in
phosphate buffer (pH 7.2; 0.5M) (2 ml per rat brain) with a hand-held glass- teflon homogenizer in an ice-cold bath. To induce
precipitation of protein, 50 μl of DMSO was
subsequently added to each brain homogenate and the organic layer (upper layer)
was separated by μg/ml
2.4. Preparation
of calibration standard solutions
A
primary stock solution of Phenytoin 1000µg/ml was
prepared by dissolving 10 mg of drug in 10 ml of DMSO. The stock solution of Phenytoin was serially diluted with DMSO to achieve working
standard solutions of concentrations of 4, 8, 12, 16 and 20 µg/ml. Blank
supernatants of the brain tissue homogenates [10]
2.5. Method
Validation
The
method was validated according to the existing information, consisting of the
study of reliability parameters: linearity, sensitivity, precision, accuracy,
recovery, specificity, selectivity.
2.5.1. Selectivity
was studied investigating the absence of endogenous interferences from extracts
of blank sample supernatant of brain homogenates of rat.
2.5.2. Calibration graphs The homogenate of
drug-free brain tissue was spiked with increasing amounts of lacosamide in the concentration range of 0.02–0.1 µg/ml.
Calibration graphs were constructed by plotting the absorbance of the drug
against the concentration.
2.5.3. The
linearity was determined at different concentrations. For inter-day, assay
was performed over 5-days separately, while the intra-day assay was performed
for 1 day by analyzing each concentration 3 times. Calibration curves were
plotted as absorbance against the concentration. From this curve the lower
limit of quantization was determined.
2.5.4. Precision
to determine intraday precision, three replicate analysis of samples were performed
on the same day. The inter day precision was accessed by analysis of samples on
five different days. Precision was expressed as % CV.
2.5.5. Accuracy was determined by performing recovery
studies by spiking different concentrations of Phenytoin
in the preanalyzed sample. All analysis was performed
in triplicate. Percentage drug recovery for analyte
with corresponding %CV was determined. [11]
2.5.6. The
limit of detection
LOD =3.3 σ / s, LOQ= 10 σ / s
Where
LOD and LOQ expressed in concentration, S is the slope of the calibration plot,
and σ the standard
deviation of the y- intercept. [12]
2.5.7. Application
of method
The
amount of Phenytoin in brain was calculated as,
V*C/M
Where,
V represents the total volume of the reconstituted extract (ml); C represents
the concentration reconstitution extract determined by UV (ng/ml)
and the M represents the weight of the rat brain in (g)[13]
3. RESULT AND DISCUSSION:
The
proposed method for determination of brain concentrations of Phenytoin is based on UV-spectrophotometer. Phenytoin dissolve much in DMSO.
Fig. No.1 : UV Spectra of Phenytoin showing λmax at 215 nm.
Fig. No. 2: Calibration curve of Phenytoin better in DMSO, which is used as solvent for
spectrophotometric method. The method was validated as per the ICH guidelines.
Table .1: Intra-day variability of Phenytoin
|
Nominal Concentration
(µg/ml) |
Mean
observed concentration (µg/ml) |
% Concentration
found (Mean±SD) |
RSD% |
|
0.02 |
0.019 |
98±0.0016 |
1.636 |
|
0.04 |
0.03762 |
98.5±0.0042 |
1.642 |
|
0.06 |
0.0494 |
98.33±0.0019 |
1.877 |
|
0.08 |
0.07162 |
89.525±0.0017 |
2.374 |
|
0.1 |
0.09866 |
98.66±0.000705 |
1.714 |
Table.2: Inter-day variability of Phenytoin
|
Nominal Concentration
(µg/ml) |
Mean
observed concentration (µg/ml) |
% Concentration
found (Mean±SD) |
RSD% |
|
0.02 |
0.021 |
98±0.00157 |
1.476 |
|
0.04 |
0.038 |
97±0.003536 |
2.052 |
|
0.06 |
0.051 |
95±0.003507 |
1.764 |
|
0.08 |
0.074 |
98.7±0.002302 |
2.110 |
|
0.1 |
0.0952 |
98.2±0.003701 |
1.947 |
Table.3: Accuracy of the method for the
determination of Phenytoin in rat brain
|
Recovery (%) of Phenytoin in spiked rat brain |
|
||||
|
Recovery level (%) |
Initial concentration (µg/ml) |
Spiked concentration (µg/ml) |
Recovery (%) |
RSD(%) |
Coefficient of variance (%) |
|
Intra day |
|
||||
|
80 |
0.05 |
0.03 |
98.48% |
0.98 |
0.876 |
|
100 |
0.05 |
0.04 |
98.55% |
0.78 |
0.801 |
|
120 |
0.05 |
0.05 |
97.27% |
1.13 |
0.765 |
|
Inter day |
|
||||
|
80 |
0.05 |
0.03 |
96.38% |
1.16 |
0.895 |
|
100 |
0.05 |
0.04 |
98.55% |
0.78 |
0.801 |
|
120 |
0.05 |
0.05 |
98.31% |
0.84 |
0.895 |
3.1. Linearity and sensitivity:
Phenytoin was showed linearity in the concentration range of 0.02-0.1 µg/ml
with correlation coefficient of 0.992. Endogenous material from homogenized
brain did not impact on the quantification of Phenytoin.
Calibration curves were obtained over the concentration range of 0.02–0.1µg/ml.
The inter-day linear regression equation of the brain homogenate was 0.998 and
for the intra-day it was 0.992.
3.2. Limits
of quantitation:
The
lowest calibration standard corresponded to the LOQ were found at 0.005 µg/ml
and for LOD at 0.002 µg/ml for Phenytoin in
supernatant of tissue homogenate.
3.3. Precision:
The
result obtained for intra-day and inter-day precision are shown in table 1 and
2 respectively. The Result of analysis showed satisfactory values of ± S.D, %
CV and % relative standard deviation which indicates that method is precise and
reproducible
3.4. Accuracy:
The
values of standard deviation were satisfactorily low and recovery was found to
be in the range of 96.38% to 98.55% which indicates reproducibility and
accuracy of this method (Table 3). This bioanalytical method has been shown to be suitable for the
determination of the concentrations of phenytoin
after 1 hour after IV administration of phenytoin to Swiss
albino mice the brain concentration was
found to be 48.67876 ng/g.
4. CONCLUSION:
All
the above results indicate that, the proposed spectrophotometric method is very
simple, accurate, rapid, precise, and sensitive for determination of Phenytoin concentration in mice brain. The developed method
provides calibration curve linear ranging from 0.02-0.1 µg/ml. The % RSD for
all parameters was found to be less than two, which revealed the validation of
new method and results obtained are in acceptance limits as per ICH guidelines.
LOD and LOQ indicate that very small quantities of drug can be estimated by
this method. Method facilitates quantitative recovery of Phenytoin
from the brain matrix without interference from the major metabolites of Phenytoin and brain endogenous matter with excellent
accurate recovery ranging from 96.8% to
102.5%. The method was found to yield better results and it is applicable to in
vivo evaluation and also birelavent in vitrio bioanalysis for
concentration of Phenytoin in mice brain.
5. ACKNOWLEDGEMENT:
The
authors like to thank the pharmacology Department of Teegala
Krishna Reddy College of Pharmacy, Hyderabad for providing facilities to carry
out this work.
6. REFERENCES:
1. Pandey S., Pandey
P., Tiwari G., Tiwari R., Bioanalysis in drug discovery and development, Pharmaceutical
methods, 2010,12,76-84.
2. Bettina K. Beyreuther,
Freitag J, Heers C, Krebsfanger N, Scharfenecker and
Thomas Stohr, Lacosamide: A
review of preclinical properties, CNS drug reviews, 2007, 13
3. Krasowski M. D., Therapeutic Drug
Monitoring of the Newer Anti-Epilepsy Medications, Pharmaceuticals
4. Greenaway, Ratnaraj
C., Sander N., Patsalos J.W., Saliva and serum lacosamide concentrations in patients with epilepsy,
EPILEPSIA, 2011, 52.94-101.
5. Beyreuther BK, Geis
C, Stohr T, Sommer C, Antihyperalgesic efficacy of lacosamide
in a rat model for muscle pain induced by TNF, Neuropharmacology,
2007-04,52.
6. Kestelyn C., Lastelle M., Higuet N., Dell’ Aiera S., Staelens L., Boulanger P., Boekens
H. Smith S., A simple HPLC–UV method for the determination of lacosamide inhuman plasma, Bioanalysis,
November 2011,3,89-93 .
7. Greenaway, Ratnaraj
C., Sander N., Patsalos J.W., A High-Performance
Liquid Chromatography Assay to Monitor the New Antiepileptic Drug Lacosamide in Patientswith Epilepsy,
Therapeutic Drug Monitoring,2010, 32,65-74.
8. Ichikawa N., Naora
K., Hirano H., Iwamoto K., Quantitation of acetazolamide in rat plasma, brain tissue and cerebrospinal
fluid by high-performance liquid chromatography, Journal of Pharmaceutical and Biomedical
Analysis, 1998, 17, 1415-1421.
9. Kim S. J., Koo T. S., Jin D., Baek M, Lee S. K., Shin D. S., Moon H., Liquid
chromatography-tandem mass spectrometry for quantification of lacosamide, an antiepileptic drug, in rat plasma and
its application to pharmacokinetic
study, Biomed Chromatography, 2011, 24, 345-350.
10. Alvesa G, Figueiredoa
L, Castel-Brancoa M, Loureirob
A, Falcaoa A, and
Caramonaa M, Simultaneous and enantioselective liquid chromatographic determination of eslicarbazepine acetate, S-licarbazepine,
Rlicarbazepine and oxcarbazepine
in mouse tissue samples using ultraviolet detection, Analytica
Chimica Acta, 16July 2007,
596-598.
11. Braggio S., Barnaby R. J., Grossi P., Cugola M., A Strategy
for validation of bioanalytical methods, Journal of
Pharmaceutical and Biomedical Analysis, 1996, 14, 375-388.
12. Castel-Branco
M. M., Almeida A. M., Falcao A. C., Macedo T. A., Caramona M. M.,
Lopez F. G., Lamotrigine analysis in blood and brain
by high-performance liquid Chromatography, Journal of Chromatography
B,2001(40), 119–127.
13. Owen A., Tette
J. N., Morgan P., Pirmohamed M., Park B. K., LC
determination of carbamazepine in murine
brain, Journal of Pharmaceutical anBiomedicalAnalysis,10 (4) 254-259,Nov.2001
Received on 05.10.2014 Accepted on 16.11.2014
© Asian Pharma
Press All Right Reserved
Asian J. Pharm. Ana. 4(4): Oct. - Dec. 2014; Page 147-150