|Liquid chromatography; Riluzole; Rat plasma; Protein
precipitation; Internal standard; Method validation; Pharmacokinetic
|Amyotrophic Lateral Sclerosis (ALS) is a progressive, fatal,
neurodegenerative disease caused by the deterioration of voluntary
muscle controlling motor neurons . Currently, Riluzole (RLZ) is the
only available, clinically approved drug for treatment of ALS . Three
mechanisms are proposed for action of RLZ: inhibition of excitatory
amino acid release, inhibition of events following stimulation of
excitatory amino acid receptors and stabilization of the inactivated
state of voltage-dependent sodium channels . RLZ offers neuroprotective
action and prolongs the life of ALS patient.
|RLZ is chemically designated as 6-(trifluoromethoxy) benzothiazol-
2-amine. It is very slightly soluble in water and is weakly basic (pKa
3.5) in nature . Post oral administration, it is rapidly absorbed from
gastro–intestinal tract and has an absolute bioavailability of about 60%
in humans . It is extensively metabolized, primarily by cytochrome
P450 1A2 .
|Due to its moderate bioavailability and plethora of adverse effects,
great potential exists to improve the pharmacokinetic properties of
RLZ. Localizing RLZ in brain tissue by brain targeted delivery can
overcome some of the adverse effects associated with RLZ. From
the literature, it is evident that a few research groups have explored
pharmacokinetic [7, 8] and formulation [9,10] approaches for
enhancing the effectiveness of RLZ in animal models. Assessment
of pharmacokinetic properties for RLZ in animal models requires
determination of plasma-concentration–time profile and such research
endeavors need a simple, reproducible and cost effective bioanalytical
|Previously reported methods for determination of RLZ in human
plasma by high-performance liquid chromatography (HPLC) [11,12]
or by liquid chromatography-coupled tandem mass spectrometry 
indicated two major drawbacks: (i) Reported HPLC methods required
higher volume of plasma sample (1 mL) for determination of RLZ and
were hence, not well suited for rat studies. (ii) The LC-MS method
reported by Chandu et al. though sensitive involves complicated
processing steps and is expensive, limiting its use in rat studies.
|Further literature survey into HPLC methods for estimation of RLZ
in pre–clinical animal models revealed a method each in rat brain 
and in mouse plasma, brain and spinal cord . Since the rat brain
matrix is significantly different in composition to rat plasma matrix,
the same method cannot be used for estimation of RLZ in rat plasma
matrix. The reported mouse plasma matrix method has long run time
(over 20 min) and lesser sensitivity (LOQ = 100 ng/mL) which is again
undesirable for routine sample analysis.
|Hence, the aim of the present study was to develop and validate a
rapid and sensitive method for the determination of RLZ in rat plasma
which could be easily applied for bioavailability enhancement or drugdrug/
food interaction studies of RLZ in rats.
|RLZ and Nebivolol (internal standard, IS) were obtained as
gift samples from Apotex Research Pvt. Ltd., Bangalore, India and
Vesta Pharmachem Pvt. Ltd., Surat, India respectively. HPLC grade
acetonitrile, methanol, potassium di-hydrogen orthophosphate
(KH2PO4) and sodium citrate were purchased from Merck laboratories,
Mumbai, India. Methyl cellulose and tween 80 were purchased from
S.D. Fine Chem Ltd., Mumbai, India. Milli–Q water purification
system (Millipore®, MA, USA) was used for obtaining high quality
HPLC grade water. Male Wistar rats were purchased from Sainath
agencies, Hyderabad, India.
|Instruments and chromatographic conditions
|The liquid chromatography system employed was Shimadzu
HPLC (Shimadzu, Japan) with solvent delivery system of two pumps
(Model LC–20AD, Prominence Liquid Chromatograph, Shimadzu,
Japan), auto injector (Model SIL–20A HT, Prominence Auto Sampler,
Shimadzu, Japan) and photo diode array (PDA) UV detector (Model
SPD–M20A, Prominence Diode Array Detector, Shimadzu, Japan).
Data collection and integration was accomplished using LC Solutions,
1.25 version software.
|An endcapped C18 reverse phase column (Luna®, 250 mm long
and 4.6 mm internal diameter, particle size 5 μm, Phenomenex, CA,
USA) equipped with a guard column of same packing material was
used for the study. A combination of methanol and phosphate buffer
(25 mM KH2PO4, pH 3.5) in the ratio of 70:30% v/v was used as the
mobile phase. Buffer was filtered through 0.22 μm Millipore filtration
membrane. The HPLC system was stabilized for 1 h at 1 mL/min flow
rate, through baseline monitoring prior to actual analysis. The RLZ
and IS were monitored at fixed wavelengths of 264 nm and 280 nm
respectively with a mobile phase flow rate of 1 mL/min in isocratic
mode. An injection volume of 50 μL was optimized for final method.
|Preparation of stocks and working standard solutions
|Primary stock solutions (1000 μg/mL) of RLZ and IS were prepared
in a volumetric flask by dissolving accurately weighed amount of RLZ
and IS in methanol separately. Working standard solutions for both
RLZ and IS were prepared by appropriately diluting the respective
stock solutions with a pre–mixed solvent containing methanol and
water in the ratio of 1:1. Working standard solution of 20 μg/mL was
prepared for IS. All stock and working standard solutions were stored
at 4°C until used for analysis.
|Preparation of calibration standard solutions
|To 90 μL of drug-free plasma, 10 μL of appropriate working
standard solution of RLZ was added to achieve standard solutions
containing 50, 100, 250, 500, 750, 1000, 2000, 3000 and 4000 ng/mL of
RLZ in plasma. These concentrations were used to construct standard
calibration curves. Of which, the concentrations of 250, 750 and 3000
ng/mL were chosen for lower quality control (LQC), medium quality
control (MQC) and higher quality control (HQC) samples respectively.
|A simple, single–step protein precipitation method was followed
for extraction of RLZ from Wistar rat plasma. To 100 μL of each plasma
sample, 10 μL of IS (20 μg/mL) was added in a 1.5 mL microfuge tube.
The mixtures were vortexed for 1 min and then subjected to protein
precipitation by adding 350 μL of acetonitrile and thorough mixing for
1 min. Samples were then centrifuged at 7826 × g at 4°C for 20 min.
A clean and clear supernatant (150 μL) was transferred to a sample
loading vial and injected into the HPLC system.
|The developed method was validated statistically as per the
guidelines given by the International Conference on Harmonization
 and United States Pharmacopoeia . Various validation
parameters of the developed method were determined using the
|Linearity, accuracy, precision and specificity
|Linearity of analytical method for RLZ was determined by developing a nine–point calibration curve in the range of 50 ng/mL
to 4000 ng/mL, analyzed in six independent runs. Calibration curves
were constructed by least-square linear regression of the peak area ratio
of analyte (RLZ) to the IS versus the nominal concentrations of RLZ
spiked to drug–free plasma samples.
|For determining the intra–day accuracy and precision, five
replicates of each quality control (QC) samples of RLZ were analyzed
twice on the same day. The inter–day accuracy and precision were
assessed by analysis of five precision and accuracy batches on three
different days across all the QC levels. In order to study the specificity
of the present method, six different lots of drug–free plasma samples
were subject to the same analytical procedure to check the potential
chromatographic interference from plasma matrix.
|Lowest limit of quantification (LLOQ) was determined as the
minimum concentration of analyte that produced relative standard
deviation (RSD) within 20% of the nominal value for both precision
and accuracy . Quantitation limit and detection limit were also
determined using standard deviation of response and slope of the
calibration curve obtained from the linear regression analysis.
|Recovery of RLZ was determined by comparing the peak area of
analyte (RLZ) obtained from plasma (extracted) samples with analytical
standard (unextracted) samples at the same nominal concentration.
Recovery study was performed across the QC levels and precision of
RLZ recovery at each level (n = 5) was determined.
|Bench top stability of RLZ in rat plasma was assessed by preparing
and storing five sets of each QC samples at room temperature and
analyzing then every 3 h up to a period of 6 h on the day of preparation.
Freeze thaw stability of RLZ in rat plasma was determined across the
QC levels for three freeze–thaw cycles. A total of four sets at each QC
level (n = 5) were prepared and one set of the prepared concentrations
was analyzed on the day of preparation (no freeze thaw cycle) and
the remaining three sets were frozen at -20°C for 24 h. All the frozen
samples were thawed and one set of QC samples was analyzed. The
remaining two sets were kept at -20°C for freezing and were analyzed
after two and three freeze thaw cycles. Long–term stability of RLZ in
rat plasma was determined across the QC samples. A total of four sets
at each QC level (n = 5) were prepared and one set of the prepared
concentrations was analyzed on the day of preparation. The remaining
three sets were frozen at -20°C. Each set of stored samples was analyzed
after 7, 15 and 30 days of sample preparation. The percentage deviation
from the mean concentrations observed at zero time was calculated in
all the stability studies.
|Male Wistar rats, weighing 180 to 220 g were used in the study.
The experimental protocol was approved by the Institutional Animal
Ethics Committee (Approval No.: IAEC–14/09–11). All animals were
fasted overnight (12 h) before dosing and continued till 4 h after
administration of test items, thereafter rat chew diet was provided ad
|A freshly prepared aqueous suspension of RLZ (containing 0.5%
methyl cellulose and 0.1% tween 80) was administered at a dose of 10
mg/kg through the oral route in rats (n = 5) in the pharmacokinetic study. Blood samples (0.5 mL) were collected from retro–orbital plexus
into the microfuge tubes containing sodium citrate as anticoagulant
(3.8% w/v) at pre dose, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12 and 16 h post dose
and kept on ice till further processing. These samples were further
harvested for plasma by centrifuging at 4°C for 10 min at 905 × g and
then stored at -20°C till further analysis. All samples were processed
according to the procedure described earlier and analyzed using the
validated HPLC method.
|Results and Discussion
|The effects of various buffers and buffer compositions with varying
pH conditions on the retention time and peak symmetry of RLZ were
investigated. Mobile phase was selected based on the short run time,
symmetric factor, sensitivity, ease of preparation and the economy of
the method for in vivo studies in rats.
|A significant effect of mobile phase composition was observed
on retention time of RLZ. Retention time of RLZ was increased from
4.1 min (drug peak merged with plasma junk) to 11.5 min (broader
peak with decreased height was observed) with a decrease in organic
phase (methanol) composition from 80% to 50% v/v in the mobile
phase. Mobile phase consisting 70% of methanol was found to provide
retention time of 6.8 ± 0.11 min for RLZ. No additional advantage
was observed in using acetonitrile in comparison with methanol on
RLZ peak properties including sensitivity and peak symmetry. Hence,
methanol was selected as an organic as it is cost effective for routine
|Peak tailing has been a major concern for ionizable basic
compounds like RLZ (with free amino group). Secondary interactions,
via an ion exchange mechanism, between protonated base (BH+)
of analyte and acidic, ionized silanol (Si–O-) groups on the surface
of silica support particles are primarily responsible for the tailing
phenomenon. These interactions and thereby tailing can be reduced
by using acidic buffers (up to pH 3) at higher buffer concentration and
choosing buffer cations that are strongly held by the silanols (18–19).
Initially, buffers like ammonium acetate and ammonium formate at
higher concentrations of up to 30 mM were tried, however, phosphate
buffer (25 mM KH2PO4) was found to be suitable in reducing the tailing
factor and in improving peak shape of RLZ.
|Final optimization was carried out by changing the pH of the
selected buffer. A decrease in buffer pH from 5.8 to 3.5 caused further
improvement on peak tailing of RLZ to the acceptable range (< 1.2).
Decreasing the pH of buffer reduced the ionization of acidic silanols
groups and thereby reducing the interaction between protonated RLZ
and silanols groups.
|In HPLC analysis, selection of proper internal standard
significantly impacts precision, accuracy and recovery of the method.
Different compounds including acyclovir, diclofenac, nebivolol and
raloxifene were tested for selection as internal standards in optimized
chromatographic conditions. Peaks of acyclovir and diclofenac showed
short retention (retention factor < 0.5) and were found to merge with
plasma components. Raloxifene showed high retention time (15.5 min)
and hence contributed to increase in total run time for each sample
that belied our objective of developing a rapid method. Only nebivolol
could be well separated from plasma junk and RLZ within short run
time of 10 min. Therefore nebivolol was selected as IS. Under the
optimal chromatographic conditions, the retention times for IS and RLZ obtained were 5.3 ± 0.09 and 6.8 ± 0.11 min respectively with good
separation (resolution > 2) between the analyte peaks.
|Specificity: Potential interference of plasma components in the
analysis of RLZ and IS was checked at two wavelengths, 264 nm and
280 nm. No significant interference, from endogenous substances
in plasma was observed either at the retention time of RLZ or at IS,
indicating specificity of the method. Results for specificity are shown in Figure 1A and Figure 1B.
|Linearity: The mean regression equation for calibration curve of
RLZ in rat plasma matrix was Y = 0.00085 (± 0.00007) X–0.03835 (±
0.0012) (r2 = 0.999), where ‘Y’ represents the ratio of peak area of RLZ
to IS, and ‘X’ represents concentration of RLZ. The calibration curve
was linear in the concentration range of 50–4000 ng/mL.
|Accuracy and precision: All three quality control samples (LQC =
250 ng/mL, MQC = 750 ng/mL and HQC = 3000 ng/mL) showed an
accuracy ranging from –1.22% to 3.25% with maximum %RSD of 4.11
across the entire QC range, establishing the accuracy of method for
RLZ estimation in rat plasma (Table 1).
|From the results obtained in intermediate precision studies, the
%RSD values for intra–day variation and inter–day variation were
found to be not more than 4.10 and 4.02 respectively (Table 1). Lower
%RSD values indicated repeatability and intermediate precision of the
|Sensitivity: Analysis of six independent samples of 50 ng/mL had shown accuracy in the range of to –3.81% to 5.52% with intra–
day and inter–day precision values of 5.83% and 6.02% respectively.
Quantification limit and detection limit determined using standard
deviation of the response and the slope of the calibration curve were
found to be 49.09 ng/mL and 16.21 ng/mL respectively. Hence, LLOQ
of 50 ng/mL was considered to be reliable, reproducible and accurate
for the proposed method. The LLOQ value obtained for RLZ in the
present study is significantly improved compared to previously
reported method (100 ng/mL) by Colovic et al. in mouse plasma.
|Extraction recovery: Recovery of RLZ from the spiked rat
plasma samples, when compared with analytical standards of same
concentration, for all the QC levels tested was in the range of 81.3% to
85.5% with %RSD less than 4.11% at each of QC levels.
|Stability: No significant degradation of RLZ in plasma was
observed under various stress conditions: bench top storage at room
temperature for 6 h, three freeze and thaw cycles and long–term storage
at -20°C for 30 days. The deviation from the zero time concentration
was found to be in the range of –1.81% to 0.84% and -2.61% to 2.05%
in bench top stability and freeze thaw stability studies respectively. In
case of long–term storage stability study, deviation from the zero day
concentration was found to be in the range of –3.90% to 2.70%. Results
obtained from stability studies are summarized in Figure 2.
|Pharmacokinetic application: The proposed analytical method
was successfully applied to study the pharmacokinetics of RLZ after
single dose oral administration of an aqueous suspension of the drug
in rats. The mean plasma concentration versus time profile of RLZ
obtained following oral administration is given in Figure 3.
|The pharmacokinetic parameters obtained from the study using
non–compartmental analysis (WinNonlin(R), 5.1 version, Pharsight
Inc., CA, USA) were area under the curve (AUC) = 8460.13 ± 560.25
h ng/mL, area under the first–moment curve (AUMC) = 55075.38 ±
3609.33 h2 ng/mL, mean retention time (MRT) = 6.51 ± 0.12 h, time to
reach the maximum plasma concentration (tmax) = 1.0 h and maximum
plasma concentration (Cmax) = 1680.46 ± 30.42 ng/mL. Samples
collected till 16 h post oral administration of the drug were analyzed
in the study, indicating sensitivity and the applicability of the proposed
method to in vivo pharmacokinetic studies of drug in rats.
|A rapid, precise, specific, sensitive and cost effective HPLC method was developed and validated for estimation of RLZ in rat plasma. The
drug was found to be stable under various processing and storage
conditions. The developed method allows high sample throughput due
to the simple procedure for sample preparation and relatively short
run time. The method was successfully employed in determining the
pharmacokinetic parameters of the drug following oral administration
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