QUANTITATIVE ANALYSIS OF TOPIRAMATE IN HUMAN ......2018/02/27 · topiramate into blank plasma (190µL) to achieve a concentration of 3000, 1500, 750, 300, 150, 75, 30 and 15 ng/mL

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Thummar et al. World Journal of Pharmacy and Pharmaceutical Sciences

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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QUANTITATIVE ANALYSIS OF TOPIRAMATE IN HUMAN ......2018/02/27 · topiramate into blank plasma (190µL) to achieve a concentration of 3000, 1500, 750, 300, 150, 75, 30 and 15 ng/mL