QUANTITATIVE ANALYSIS OF TOPIRAMATE IN HUMAN ......2018/02/27 · topiramate into blank plasma...
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QUANTITATIVE ANALYSIS OF TOPIRAMATE IN HUMAN PLASMA
USING LC-MS/MS AND ITS APPLICATION TO PHARMACOKINETIC
STUDY
Kashyap Thummar1*
, Darshan Jethva1 and Navin Sheth
2
1Department of Pharmaceutical Sciences, Saurashtra University, Rajkot - 360005, Gujarat,
India.
2Gujarat Technological University, Ahmedabad - 382424, Gujarat, India.
ABSTRACT
A simple, sensitive and reliable liquid chromatography-tandem mass
spectrometry (LC-MS/MS) method was developed for the
quantification of topiramate in human plasma. The liquid-liquid
extraction was used to extract topiramate from human plasma.
Chromatographic separation was achieved on Gemini C18 (150 mm x
4.6 mm, 5µm) column with the mobile phase composed of acetonitrile:
2 mM ammonium acetate (85:15, v/v) at a flow-rate of 0.5 mL/min.
The method was validated over the linearity range 15 – 3000 ng/mL
with 0.2 mL of human plasma using niclosamide as an internal
standard (IS). The precursor to product ion transition was monitored at
m/z 338.20 → 78.20 and m/z 325.20 → 171.20 for topiramate and IS
in negative mode, respectively. This method confirmed precision, accuracy and extraction
recoveries obtained for topiramate were consistent and reproducible. Topiramate was found
to be stable throughout the freeze-thaw cycles, bench-top and post-operative stability studies.
The method was successfully applied to epileptic patients to monitor the plasma drug
concentration versus time profile. The inter variability in pharmacokinetic parameters shows
the need for individual monitoring of topiramate plasma profile by the accurate, specific and
sensitive bioanalytical method.
KEYWORDS: Bioanalytical method; Human plasma; LC-MS/MS; Pharmacokinetic study;
Topiramate.
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 7.421
Volume 7, Issue 5, 1016-1032 Research Article ISSN 2278 – 4357
*Corresponding Author
Kashyap Thummar
Department of
Pharmaceutical Sciences,
Saurashtra University,
Rajkot - 360005, Gujarat,
India.
Article Received on
27 Feb. 2018,
Revised on 20 March 2018,
Accepted on 09 April 2018,
DOI: 10.20959/wjpps20185-11511
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1. INTRODUCTION
Epilepsy is a chronic non-communicable disorder of the brain that affects people of all ages.
Approximately 50 million people worldwide have epilepsy, making it one of the most
common neurological diseases globally.[1]
Topiramate (2,3:4,5-Bis-O-(1-methylethylidene)-
beta-D-fructopyranose sulfamate) is a second generation anti-epileptic drug widely used drug
alone or in combination for the treatment of epilepsy. It is useful for several types of partial-
onset and generalized-onset seizures and is therefore considered a broad-spectrum agent.[2]
It
is also effective as a prophylactic against a migraine headache.[3]
Topiramate is rapidly
absorbed after oral administration, shows little (10-20%) binding to plasma protein. It is
mainly excreted unchanged in the urine and by diverse metabolic pathways. Food slows the
absorption but there is no significant effect.[4-5]
Topiramate level monitoring in blood has
been playing important role in treatment of epilepsy to determine the optimum therapeutic
dose for an individual patient.
The topiramate does not have UV-Vis absorption or emit fluorescence and could not quantify
by readily accessible techniques, nevertheless, a variety of analytical methods have been
reported for quantitative determination of topiramate. It often employs derivatization methods
followed by separation and/or detection. The efforts have been established to quantify
topiramate by high performance liquid chromatography (HPLC) combined with different
detector viz. indirect UV[6]
, fluorescence.[7-12]
, refractive index[13]
and chemiluminescent
nitrogen detection[14]
in pharmaceutical formulations as well as from biological fluids.
Further, spectrofluorimetric method[15]
, gas chromatography-mass spectrometry (GC-MS)[16]
and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were also
reported to investigate topiramate in various biological matrices such as in human plasma[17-
22], serum
[23], dried blood spots
[24] and in combination with other drugs in human plasma
[25-28]
and serum[29-30]
also. Moreover, Yin et al. discussed the comparative assessment of reported
methods thoroughly for topiramate and sets many reported methods to have relatively long
analytical run time, large sample volumes, and complex sample preparation. Additionally,
Yin et al. were developed LC-MS/MS method to determine anti-epileptic drugs in human
plasma using protein precipitation extraction technique (PPT).[28]
Studies have shown that
PPT could not remove phospholipid as compared to liquid-liquid extraction (LLE) presented
in plasma which may be affecting the results. Hence, sometimes PPT was considered as less
clean extraction or crude extraction technology from plasma.[31]
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This method was demonstrated to provide sensitive, precise and accurate LC-MS/MS method
for the quantification of topiramate in human plasma. This method allows simple and fast
sample preparation using LLE technique and shorter runtime. The method was applied to a
four epileptic patient to monitor topiramate concentration in plasma and to study
pharmacokinetic behavior.
2. MATERIAL AND METHODS
2.1. Chemical and reagents
Topiramate and niclosamide (IS) were the generous gifts from Torrent Pharmaceutical Ltd.,
and Ami Lifescience Pvt. Ltd., India, respectively. HPLC grade acetonitrile, methanol and
tert-butyl methyl ether were purchased from Spectrochem Pvt. Ltd., India. Ultrapure water
was used throughout the study from MilliQ-Elix system, Millipore Pvt. Ltd., India. All other
chemicals used in this study were analytical grade and used without further purification. The
control human plasma with K3EDTA anticoagulant was obtained from Rajkot voluntary
blood bank and research center, Rajkot, Gujarat, India.
2.2. Instrumentation and analytical conditions
The LC-MS/MS system consisted of a LC-20AD solvent delivery system, a DGU-20A5R
vacuum degasser, SIL-20AC autosampler and CTO-20AC thermostated column oven
connected by an electrospray ionization (ESI) interface and coupled with a triple quadrupole
mass spectrometer LCMS-8030 (Shimadzu Corporation, Kyoto, Japan). Data acquisition and
processing were performed using Lab solution software (version 5.53) from Shimadzu
Corporation, Japan.
The chromatographic separation was carried out on a Gemini C18 (150 x 4.6 mm, 5μm)
analytical column placed in thermostated column oven maintained at 40 oC. The mobile
phase consisted of acetonitrile: 2mM ammonium acetate in the proportion of 85:15, v/v at a
flow rate of 0.500 mL/min. The autosampler temperature was kept at 4oC and 10 µL sample
volume was injected into the system. Analytical run time was 7 min.
In mass, the ionization and detection were performed by infusing a solution containing 500
ng/mL of topiramate and IS. The operating conditions were set as follows; the interface
temperature and ion spray voltage fixed at 350 °C and 4.5 kV respectively. Nitrogen was
used as nebulizing gas (3 L/min) and drying gas (15 L/min). With argon as collision gas,
multiple reaction monitoring (MRM) mode was applied to the ionic transition of m/z 338.20
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→ 78.20 for topiramate and m/z 325.20 → 171.20 for IS in negative ionization mode. The
optimized collision energies 30eV were used for both. The dwell time was set to 100 ms.
2.3. Preparation of Standard and quality control (QC) Samples
The stock solutions of topiramate (1000 µg/mL) and IS (1000 µg/mL) were prepared in
methanol. The stock solution was further serially diluted using a mixture of methanol and
water in the ratio of (50:50, v/v) to get series of working standard solution having a
concentration of 60000, 30000, 15000, 6000, 3000, 1500, 600 and 300 ng/mL. From that
calibration curves were prepared by 5% spiking (10µL) of the working standard solutions of
topiramate into blank plasma (190µL) to achieve a concentration of 3000, 1500, 750, 300,
150, 75, 30 and 15 ng/mL for topiramate. The quality control samples (QCs) were prepared in
a similar manner at four concentration level, viz. 2700 ng/mL (high, HQC), 225 ng/mL
(middle, MQC), 45 ng/mL (low, LQC) and 15 ng/mL (lower limit of Quantification, LLOQ
QC) for topiramate; A working IS solution was prepared in same way to get final
concentration of 500 ng/mL. All samples were stored at 2 – 8oC until analysis.
2.4. Plasma sample preparation
To 200 µL of spiked plasma, add 50 µL of internal standard and vortex for 30 sec in 2.5 mL
polypropylene vial. A 50 µL of extraction buffer (0.1N HCl) was added and vortex for 1 min.
Subsequently, 1.3 mL of liquid extraction solvent (tert-butyl methyl ether) was added to
above solution and vortex for 3 mins. The resultant mixture was then centrifuged at 4000 rpm
at 10oC for 10 mins, the organic phase was transferred to a vial and evaporated to dryness at
40oC under a gentle stream of nitrogen. The dried samples were reconstituted with 100 µL of
mobile phase as a reconstitution solvent and a 10µL sample were injected into the
chromatographic system.
2.5. Bioanalytical method validation
The method was validated based on recommendation of the United States Food and Drug
Administration (USFDA) guidelines.[32]
2.5.1.Specificity
The specificity of the method was evaluated by screening ten different batches of blank
human plasma, (seven were K3EDTA and one each of lipidemic, haemolysed and heparinized
plasma) to investigate the interferences at the signal of topiramate and IS [33]
. Aliquots of
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plasma samples were used to prepare LLOQ and blank samples. The baseline noise should be
< 20% of analyte response at this concentration level.
2.5.2. Linearity, accuracy and precision
The linearity of the method was determined by analysis of an eight-point calibration curve
over the concentration range of 15-3000 ng/mL of topiramate. Each calibration curve (n = 3)
was analyzed individually by least square weighted linear regression method (1/x2) of peak
area ratios of topiramate to IS (y) versus actual concentration (x). Slope (m) and intercept (c),
were calculated for each standard curve. The regression equation (y = mx + c) for the
calibration curve was used to back-calculate the measured concentrations at each standard as
well as QC level. A correlation coefficient (r2) value of greater than 0.990 was desirable for
all calibration curves.
The intra and inter assay precision and accuracy were determined by analyzing a set of QC
samples (n = 6) at LLOQ QC, LQC, MQC and HQC level (15, 45, 225 and 2700 ng/mL,
respectively) on the same day and on three consecutive days respectively. The percentage
relative standard deviation (%RSD) and percent deviation from the nominal concentration (%
Bias) serves as a measure of precision and accuracy, respectively. The criteria for
acceptability of the data included precision and accuracy were within ±15% of nominal value,
except for LLOQ, where it should not exceed ± 20% of precision and accuracy as well.
2.5.3. Recovery and matrix effect
The extraction recovery and matrix effect for topiramate were performed in six replicates of
LQC, MQC and HQC samples (45, 225 and 2700 ng/mL). The recovery of the topiramate
after liquid-liquid extraction was evaluated by comparing peak area obtained from pre (A)
and post (B) spiked plasma extracted samples at equivalent concentrations. The percentage
recovery was measured by . The matrix effect due to plasma matrix was
assessed by comparing peak area of the post spiked samples (B) to those of pure standard
solutions in mobile phase at the same concentrations (C). The matrix effect (%) was
calculated by . The extraction recovery and matrix effect of IS were evaluated
at the working concentration (500 ng/mL) in the same manner. The %RSD of recovery and
matrix effect at each concentration should be less than 15%.
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2.5.4. Dilution integrity
Dilution integrity was investigated to ensure that samples could be diluted with blank matrix
without affecting the final concentration in case if study samples go beyond the validated
calibration range of method. To demonstrate the dilution integrity of topiramate, pre-
determined aliquots were diluted with human plasma (1:2 and 1:10) to form diluted quality
control samples (DQCs) having a concentration of DQC (1/2) 1200ng/mL and DQC (1/10)
2700 ng/mL. The back-calculated standard concentrations had to comply both precision of
≤15% and accuracy of 100 ± 15% similar to other QC samples.
2.5.5. Stability
The stabilities in human plasma were tested by analyzing six replicates of low and high
matrix matched QC samples (45, 2700 ng/mL) in different conditions. The bench top stability
(6 h at ambient temperature), freeze-thaw stability (-20 ± 5°C), Autosampler (wet extract)
stability (at 4oC for 24 h), dry extract stability (2 - 8°C for 24 h.), and long term stability (-20
± 5°C for 28 days) were determined. The concentration of stability samples and freshly
prepared samples were calculated and stability was shown as the percentage mean stability in
calculated concentration. If the % RSD of mean stability was within 15%, the assay was
considered stable.
2.6 Application of the method
The main objective of this clinical study was to prove the applicability of the analytical
technique. Prior to the start, the study protocol and statement of informed consent were
approved by human ethics committee of Department of Pharmaceutical Sciences affiliated to
Saurashtra University (Rajkot, Gujarat, India). Meanwhile, the clinical study was performed
according to the ethical values of the Declaration of Helsinki. An experienced specialist
neuro-physicians of Wellcare hospital (Rajkot, Gujarat, India) carried out the diagnosis of the
disease and prescribed appropriate drug therapy for its patients. In this manner, total four
patients under topiramate therapy were enrolled for the experimental monitoring. The routine
dose of 50 mg of topiramate was set for examination in a day. Approximately 3ml of blood
sample were drawn into an EDTA tube at 0, 0.5, 1, 2, 3, 4, 12 and 24 h after administration of
50 mg topiramate tablet. The plasma samples were prepared by centrifuging the blood
samples at 3000 rpm, for 10 min at Wellcare Pathology Laboratory (Rajkot, Gujarat, India).
The plasma samples were stored at -20 oC prior to sample processing.
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3. RESULTS
3.1 Optimization of mass spectrometric condition
The topiramate and IS were analyzed firstly by direct infusion of the individual standard
solution. Both positive and negative ionization modes were inspected for the detection of
topiramate and IS, and negative ionization mode gave more efficiently ionized and therefore
employed. In this manner, the molecular ion [M-H]- of topiramate and IS was detected at
338.2 m/z, and 325.20 m/z respectively. The collision-induced (30 eV for topiramate and IS)
dissociation at 78.2 m/z due to the stabilized product ion of H2NO2S from topiramate,
whereas 171.20 m/z due to stabilized product ion of C6H4ClN2O2•. The corresponding mass
spectrum as shown in Figure 1.
3.2 Optimization of sample preparation
One of the key central steps in the development of the bioanalytical method is sample
preparation. Sample preparation procedure should be rapid, easy and should require the least
amount of reagents with maximum recovery of analytes. In this regard, we used LLE method
in such a way that the sample preparation took shorten processing time and to acquire desired
recoveries of the topiramate and IS. Different organic solvents such as dichloromethane,
diethyl ether, n-hexane, ethyl acetate, tert-butyl methyl ether and several buffers such as 0.1
N HCl, 0.1 N NaOH, etc were used in trials. Among them, tert-butyl methyl ether and 0.1 N
HCl showed maximum and reproducible recovery. There was no interference from any
endogenous or exogenous plasma matrix and IS did not alter topiramate recovery, sensitivity
and/or ion suppression as well.
3.3 Optimization of chromatographic condition
The chromatographic conditions were optimized to look for better sensitivity, peak shape and
chromatographic run time. The selection of mobile phase was performed taking into account
the symmetric peak shape with a shorter run time that further leads to low consumption of
organic solvent altogether making the method cost-effective. In this study, we tried Gemini-
C18, Kromasil-C18 with various organic solvent and buffer solutions for mobile phases, such
as methanol, acetonitrile, 0.1% formic acid, 2mM ammonium acetate and ammonium
formate. The satisfactory results were obtained in Gemini C18 (150 x 4.6 mm, 5μm)
analytical column with the several combinations showed that acetonitrile: 2mM ammonium
acetate buffer (85:15, v/v) serves the desired purpose with utmost effectiveness.
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3.4 Method validation
3.4.1 Selectivity
Figure 2 shows the representative mass chromatograms of blank plasma for topiramate and
IS, standard zero (IS) and LLOQ sample were presented. No significant interference in the
blank plasma traces was observed from endogenous substances in drug free human plasma at
the retention time of topiramate and/or IS. The retention times were 3.64 and 4.94 min for
topiramate and IS respectively.
3.4.2 Linearity, accuracy and precision
The chromatographic responses were found linear over the concentration range of 15 – 3000
ng/mL in human plasma. The correlation coefficient, r2 was consistently equal or greater than
0.995 during the course of validation. The calibration curve samples were within ± 15% of
the theoretical value. The intra and inter-assay precision and accuracy were assessed by
analyzing six replicate at each QC level. Concentrations were back-calculated from the
calibration curve. The method was reliable and reproducible since the intra and inter-assay
precision (% RSD) was less than 9.75% and 8.82%, respectively. On the other hand, intra and
inter-assay accuracy (% Bias) was within 6.19% and 7.66%, respectively. Table 1 showed
that all values of accuracy and precision fell within the limits as acceptable.
3.4.3 Recovery and matrix effect
The topiramate and IS were recovered by LLE from human plasma. The % recoveries of
topiramate were ranged from 89.12 - 93.09%. The recovery of IS was achieved to be 92.81 ±
5.42%. Table 2 showed that the recovery of topiramate and IS were high and consistently
precise and reproducible. The mean value of matrix effect of topiramate was varied from
91.59% - 96.16% with acceptable %RSD value. The data represented in Table 2 indicates
that no endogenous substance significantly influenced ion suppression of topiramate and IS
as well in this analytical method and allowed us to conclude that our method is able to
quantify topiramate in human plasma.
3.4.4 Dilution integrity
Precision (%RSD) values for reliability for 1/2nd
and 1/10th
dilutions were 9.61 and 6.36%,
while the accuracy was 102.82 and 97.09% for topiramate, respectively. Results were within
the acceptance limit of 15% for precision and 85 – 115% for accuracy as shown in Table 3.
This parameter was especially important to prove that the quantification of topiramate was
also possible for the out of range sample. In this study, we faced the same for one patient
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(Patient A) who’s the plasma concentration of topiramate was significantly higher, in that
case, the sample was diluted 2 times and thereafter processed and analyzed.
3.4.5 Stability
Table 4 summarizes the results of the bench-top stability, freeze-thaw stability, dry extract
stability, autosampler stability and long term stability in human plasma as the percentage
mean stability of the calculated vs. theoretical concentration. No significant deviations were
observed compared to theoretical concentration, indicating that topiramate was stable under
all tested conditions. Therefore, the method has been proved to be applicable for routine
analysis.
3.5 Application of the method
The validated method was applied to monitoring the plasma concentration of topiramate from
the four epileptic patients. The calibrations, QCs and real samples were run and analyzed
successfully on the same batch. The mean plasma concentration versus time profile is
presented in Figure 3. The pharmacokinetic parameters such as peak plasma concentration
(Cmax), time required to achieve maximum plasma concentration (Tmax), half-life (t1/2), Area
under curve (AUC), mean residence time (MRT), Elimination rate (Lz) and clearance (CL) of
topiramate were calculated by pksolver tool in excel sheet and summarized in Table 5.
Figure 1: Mass spectrum of (a) topiramate and (b) IS, precursor to product ion with its
possible fragmentation pattern.
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Figure 2: Mass chromatogram of (a) blank plasma (b) Zero standard (IS) (c) LLOQ
sample of topiramate (15 ng/mL) (d) ULOQ sample of topiramate (3000 ng/mL).
Figure 3: Plasma concentration of topiramate versus time profile of four epileptic
patients.
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Table 1: Accuracy and precision values of topiramate in human plasma.
Nominal conc. (ng/mL) Found (mean ± S.D.) (ng/mL) % RSD % Bias
Intra-assay (n=6)
15 14.62 ± 1.42 9.75 2.56
45 42.22 ± 2.41 5.71 6.19
225 217.50 ± 12.94 5.95 3.33
2700 2546.15 ± 131.08 5.15 5.70
Inter-assay (n=18)
15 14.87 ± 1.31 8.82 0.85
45 44.07 ± 3.65 8.28 2.06
225 211.72 ± 11.89 5.62 5.90
2700 2493.25 ± 126.80 5.09 7.66
Table 2: Extraction recovery and matrix effect of topiramate in human plasma.
Analytes Spiked conc.
(ng/mL)
Recovery
(% mean ± SD) % RSD
Matrix effect
(% mean ± SD) % RSD
Topiramate
45 89.12 ± 4.55 5.10 91.59 ± 3.53 3.86
225 92.24 ± 3.38 3.66 96.16 ± 2.28 2.37
2700 93.09 ± 4.07 4.37 92.19 ± 2.10 2.28
IS 500 92.81 ± 5.42 5.83 84.81 ± 4.28 5.05
Table 3: Dilution integrity of topiramate in human plasma.
DQC (1/2) DQC (1/10)
Dilution concentration (ng/mL) 1200 2700
Mean ±SD (n=6) (ng/mL) 1233.83 ± 118.58 2621.50 ± 166.76
% Mean Accuracy 102.82 97.09
% RSD 9.61 6.36
Table 4: Stability data of topiramate under different storage conditions.
Stability conditions
LQC (45 ng/mL) HQC (2700 ng/mL)
Mean ±SD
(n = 6)
%
RSD
% Mean
stability
Mean ±SD
(n = 6)
%
RSD
% Mean
stability
Bench top stability for 6 h
(at ambient temperature) 45.42 ± 3.33 7.34 100.93
2609.48 ±
69.65 2.67 96.65
Freeze and Thaw stability
at -20 oC (5 cycle)
43.12 ± 3.05 7.08 95.83 2588.33 ±
91.38 3.53 95.86
Dry extract stability at -20 oC for 24 h
46.03 ± 2.50 5.43 102.30 2623.33 ±
86.58 3.30 97.16
Auto sampler stability 4 oC
for 24 h 44.27 ± 1.94 4.39 98.37
2604.93 ±
34.52 1.33 96.48
Long term stability at -20 oC for 28 days
42.71 ± 3.28 3.28 94.90 2535.00 ±
83.07 3.28 93.89
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Table 5: Pharmacokinetic parameter of topiramate after oral administration 50 mg to 4
epileptic patients.
Subject
Pharmacokinetic parameter
Tmax Cmax t1/2 AUC0 → 24 MRT0 → 24 Lz CL
(h) (µg/mL) (h) (µg*h/mL) (h) (h-1
) (L/h)
Patient A 2 5.98 102.03 78.64 11.21 0.68 0.11
Patient B 3 1.08 49.97 19.91 11.54 1.39 0.68
Patient C 0.5 1.92 23.39 25.09 10.10 2.96 0.97
Patient D 1 2.03 67.77 40.94 11.40 1.02 0.26
A – female, 40 year old, 65 kg., B – female, 29 year old, 75 kg., C – male, 17 year old, 60
kg., D – female, 50 year old, 66kg.
4. DISCUSSION
The topiramate and internal standard were separated on analytical column C18 (150 x 4.6
mm, 5μm) with acetonitrile: 2mM ammonium acetate (85:15, v/v) as a mobile phase. The
ionization efficiency of ESI is higher in case of acetonitrile and ammonium acetate because
of the properties like low viscosity and volatile salts, respectively. The mass spectrometric
conditions were optimized to increase sensitivity and specificity of the method. The MS/MS
conditions were optimized to obtain the best possible sensitivity. Multiple reaction
monitoring mode was used to monitor precursor to product ion, (338.2 78.2 m/z for
topiramate and 325.20 171.20 m/z for IS), which could reduce interference and enhance
selectivity. The method was very sensitive to quantitate 15 ng/mL at LLOQ level which was
enough for topiramate estimation. In this study, we follow liquid-liquid extraction for the
high throughput recovery of topiramate from human plasma which has advantages to get
cleaner sample than protein precipitation technique and also cost-effective than solid phase
extraction.
Further, the developed method was accurate and precise, the values of this fell within the
limits as acceptable. The high efficient and reproducible liquid-liquid extraction with no
matrix effect altogether allowed us to conclude that our method is able to quantify topiramate
in human plasma. It is important to have dilution integrity in a method to represent the wide
applicability of the method towards the real samples. The topiramate was stable over a
variety of stated conditions and it was said to be a rugged method.
This chromatographic method was also checked for its applicability to real samples. The
plasma concentration of topiramate with respect to time was helped to calculate the
pharmacokinetic parameters. The inter-variability in the pharmacokinetic parameters shows
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the need for individual monitoring of topiramate in epileptic patients and dose-related adverse
effects may be obviated in most patients by dose optimization.
5. CONCLUSIONS
A novel LC-MS/MS method for the estimation of topiramate from human plasma and its
pharmacokinetic application was developed and validated according to the USFDA
guidelines. This is significant because the estimation of topiramate is quite difficult because it
does not have UV-Vis absorption or emit fluorescence, which is unfavorable for
determination by readily accessible techniques. Therefore, the LC-MS/MS method was
developed for topiramate in human plasma, prove to have good specificity, improved
sensitivity and reproducible linearity. Moreover, this method offers significant advantages
over those previously reported methods in the biologic matrix, in terms of highly efficient
liquid-liquid extraction method. In addition, the absence of matrix effect and stable under
routine handling and processing conditions leads to a reproducible and rugged method for
quantification of topiramate in human plasma. The method was successfully applied to the
real samples. The plasma concentration profile and its pharmacokinetic parameters of
individuals were greatly useful for dosage optimization. Thus, the developed LC-MS/MS
method was found to be reliable for an intended application.
ACKNOWLEDGEMENT
The author wishes to thank, University Grant Commission (UGC), Government of India for
awarding BSR meritorious fellowship for doctoral research. The author would also like to
thank Dr. Bipin Bhimani, Dr. Gaurav Dave and whole Wellcare hospital team for their
continuous support for providing necessary facilities to the patient during blood sample
collection.
Compliance with ethical standards
The study has been approved by Institutional Ethics Committee, Department of
Pharmaceutical Sciences, Saurashtra University, India. And it has been performed in
accordance with the ethical standards of the guideline. (Approved protocol no.
SUDPS/ETHICLIN/03/2014/14).
Conflict of interest
The authors declare no conflict of interest.
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Informed Consent
A written consent was obtained from all the individuals involved in the study.
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