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Determination of Rizatriptan in Human Plasma by Liquid Chromatography
Stable Isotope Dilution Electrospray MS–MS for Application in Bioequivalence
Study
Article in Chromatographia · October 2011
DOI: 10.1007/s10337-011-2110-7
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Chromatographia
DOI 10.1007/s10337-011-2110-7
ORIGINAL
Determination of Rizatriptan in Human Plasma by Liquid
Chromatography Stable Isotope Dilution Electrospray MS–MS
for Application in Bioequivalence Study
Ramakotaiah Mogili • Kanchanamala Kanala
Balasekhara R. Challa • Babu R. Chandu •
Chandrasekhar K. Bannoth
•
Received: 28 November 2010 / Revised: 18 June 2011 / Accepted: 24 June 2011
Ó Springer-Verlag 2011
Abstract A simple, sensitive, selective, rapid, rugged,
reproducible and specific liquid chromatography–tandem
mass spectrometry (LC–MS/MS) method was used for
quantitative estimation of rizatriptan (RZ) in human plasma
using rizatriptan-d6 (RZD6) as internal standard (IS).
Chromatographic separation was performed on Ascentis
Express RP Amide C18, 50 9 4.6 mm, 2.7 lm column with
isocratic mobile phase composed of 10 mM ammonium
formate:acetonitrile (20:80 v/v) at flow rate of 0.5 mL min-1.
RZ and RZD6 were detected with proton adducts at
m/z (amu) 270.2 ? 201.2 and 276.1 ? 207.1, respectively,
in multiple reaction monitoring (MRM) positive mode.
Liquid–liquid extraction was used and validated over a linear
concentration range of 0.1–100.0 ng mL-1 with correlation
coefficient r2 C 0.9981. The limit of quantification (LOQ)
and limit of detection (LOD) were found to be 0.1 ng mL-1
and 12.5 fg, respectively. Intra- and inter-day precision were
within 1.7–3.1% and 2.8–3.7%, and accuracy within 96.0–
101.7% and 99.7–101.4% for RZ. Drug was found to be
R. Mogili K. Kanala (&) C. K. Bannoth
Jawaharlal Nehru Technological University,
Anantapur 515002, AP, India
e-mail: kanchanareddy1@gmail.com
R. Mogili (&)
Siddhartha Institute of Pharmaceutical Sciences,
Jonnalagadda, Narasaraopet, Guntur 522601, India
e-mail: mrk.pharma@gmail.com;
sekharareddy2121@gmail.com
B. R. Challa
Nirmala College of Pharmacy, Kadapa 516002, AP, India
B. R. Chandu
Donbosco P.G. College of Pharmacy,
Pulladigunta, Guntur, AP, India
stable throughout three freeze–thaw cycles. The method was
successfully employed for analysis of plasma samples following oral administration of RZ (10 mg) in 25 healthy
Indian male human volunteers under fasting conditions.
Keywords Column liquid chromatography Mass
spectrometry Rizatriptan Human plasma Bioequivalence
Introduction
Rizatriptan benzoate (RZ) (N,N-dimethyl-2-[5-(1,2,4-triazole-1-ylmethyl)-1H-indol-3-yl] ethanamine monobenzoate)
is a triptan drug and selective 5-hydroxy triptamine 1B/1D
(5-HT1B/1D) receptor agonist [1]. RZ binds with high
affinity to human cloned 5-HT1B and 5-HT1D receptors,
showing weak affinity for other 5-HT1 receptor subtypes
(5-HT1A, 5-HT1E, and 5-HT1F) and the 5-HT7 receptor,
but has no significant activity at 5-HT2, 5-HT3, a- and
b-adrenergic, dopaminergic, histaminergic, muscarinic or
benzodiazepine receptors. Current theories on the aetiology
of headache suggest that symptoms are due to local cranial
vasodilatation and/or to the release of vasoactive and proinflammatory peptides from sensory nerve endings in an
activated trigeminal system. After oral doses, peak plasma
RZ concentrations are obtained about 1–1.5 h depending
on the formulation. Bioavailability is about 40–45%. Food
may delay the peak plasma concentrations of the tablet
formulation by about 1 h. Plasma protein binding is low
(14%). RZ metabolizes primarily to the inactive indole
acetic acid derivative. The active metabolite N-monodesmethyl rizatriptan is formed to a minor degree, and
other mono metabolites are also produced: about 14% as
the indole acetic acid metabolite and 1% as N-mono-
123
R. Mogili et al.
desmethyl rizatriptan. The plasma half-life is about 2–3 h
[2–8].
A number of analytical methods have been developed
for quantification of RZ in many biological matrices, e.g.
human plasma [9–17], dog plasma [18] and rat plasma
[19], and in pharmaceutical formulations [20–22]. These
methods include LC–MS [9–12, 18, 20] and high-performance liquid chromatography (HPLC) with ultraviolet
(UV) detection [13–17, 19, 21]. Among these, the best
quantification results achieved for RZ in human plasma
were by the LC–MS/MS method [10, 11, 12].
The reported methods do not show high recovery and
low matrix effect. Furthermore, comparison of rizatriptan
with deuterium-labelled internal standard has not yet been
reported.
The purpose of this investigation is to develop a simple,
sensitive, selective, rapid, rugged, reproducible and highrecovery LC–MS/MS method for quantitative estimation of
RZ using an internal standard labelled with deuterium
isotope. It is also expected that this method will provide an
efficient solution for pharmacokinetic, bioavailability and
bioequivalence studies of RZ.
Experimental
Instrumentation and Chromatographic Conditions
Mass-spectrometric detection was performed on an API
4000 triple-quadrupole instrument (ABI-SCIEX, Toronto,
Canada). HPLC was performed using a 1200 series device
(Agilent Technologies, Waldbronn, Germany). Turbo ion
spray positive mode with unit resolution and MRM were
used for detection. For RZ, [MH]? m/z (amu) 270.2 was
monitored as the precursor ion, and a fragment at m/z (amu)
201.2 was chosen as the product ion. As internal standard,
[MH]? m/z (amu) 276.1 was monitored as the precursor ion,
and a fragment at m/z (amu) 207.1 was monitored as the
product ion (Fig. 2a–d). Mass parameters were optimized as
source temperature of 450 °C, nebulizer gas at 35 psi, heater
gas at 35 psi, curtain gas at 30 psi, collisionally activated
dissociation (CAD) gas (nitrogen) at 4 psi, ion spray voltage
of 5,500 V, entrance potential of 10 V, declustering potential of 40 V, collision energy of 16 V for for both RZ and
RZD6 and collision cell exit potential of l6 V for RZ and 8 V
for RZD6. Ascentis Express RP Amide, 50 9 4.6 mm,
2.7 lm was selected as the analytical column. Column
temperature was set at 40 °C. Mobile phase composition was
10 mM ammonium formate:acetonitrile (20:80 v/v). Source
flow rate was 500 lL min-1 without split. Injection volume
was 5 lL. RZ and RZD6 were eluted at 0.92 ± 0.2 min,
with total runtime of 3 min for each sample.
Chemicals and Reagents
Rizatriptan benzoate (RZ, 99.20% purity) was purchased
from TLC Pharmachem (Ontario, Canada), and rizatriptand6 benzoate (RZD6, 99.50% purity) was obtained from
Synfine Research (Ontario, Canada) (Fig. 1). HPLC-grade
methanol and acetonitrile were purchased from Jt. Baker
Mallinckrodt Baker, Inc. (Phillipsburg, NJ, USA).
Ammonium formate (reagent grade) was purchased from
Merck Limited (Worli, Mumbai). Anhydrous sodium carbonate and methyl t-butyl ether were purchased from
Merck Speciality Private Limited (Worli, Mumbai).
Human plasma (K2EDTA) was obtained from Doctors
Pathological Lab, Hyderabad, India. Ultrapure water from
Milli-Q system (Millipore, Bedford, MA, USA) was used
through the study. All other chemicals in this study were of
analytical grade.
Fig. 1 Chemical structures of
a rizatriptan and b rizatriptan-d6
(IS)
123
Preparation of Standards, Calibration and Quality
Control (QC) Samples
Standard stock solutions of RZ (100.0 lg mL-1) and
RZD6 (100.0 lg mL-1) were prepared in methanol. IS
spiking solutions (30.0 ng mL-1) were prepared in 50%
methanol from RZD6 standard stock solution. Standard
stock solutions and IS spiking solutions were stored in
refrigerator conditions (2–8 °C) until analysis. Standard
stock solution of RZ was added to drug-free human plasma
to obtain RZ concentration levels of 0.1, 0.2, 1.0, 5.0, 10.0,
20.0, 40.0, 60.0, 80.0 and 100.0 ng mL-1 for analytical
standards and 0.1, 0.3, 30.0, 70.0 ng mL-1 [lower limit of
quantification (LLOQ), low quality control (LQC), medium
quality control (MQC), and high quality control (HQC)] for
quality control standards and stored in a freezer below
Determination of Rizatriptan in Human Plasma by Liquid Chromatography
Fig. 2 Mass spectra of: a rizatriptan Q1 scan and b fragmentation of rizatriptan, c rizatriptan-d6 Q1 scan and d fragmentation of rizatriptan-d6
-30 °C until analysis. Aqueous standards were prepared in
reconstitution solution [10 mM ammonium formate:acetonitrile (20:80 v/v)] and stored in a refrigerator (2–8 °C) for
validation experiments until analysis.
Sample Preparation
Liquid–liquid extraction (LLE) was used to isolate RZ and
RZD6 from human plasma. Fifty microlitres of RZD6
(30.0 ng mL-1) and 100 lL of respective plasma concentrations were added to polypropylene tubes and vortexed
briefly. This was followed by addition of 100 lL 0.5 N
sodium carbonate solution and 2.5 mL methyl t-butyl ether
into each tube and vortexing for 10 min. All samples were
centrifuged at 4,000g at 20 °C for 10 min, and then the
supernatant from each sample was transferred to respective
polypropylene tubes. Samples were evaporated to dryness
under nitrogen at 40 °C. Finally, samples were reconstituted with 400 lL reconstitution solution [10 mM ammonium formate:acetonitrile (20:80 v/v)] and vortexed briefly.
From this, 5 lL of each sample was injected into the HPLC
system connected to the mass spectrometer.
Linearity, Precision and Accuracy
Analytical curves were constructed using values ranging
from 0.1 to 100.0 ng mL-1 for RZ in human plasma.
Calibration curves were obtained by 1/conc2 linear
regression analysis. Calibration curves were plotted against
the ratio of instrument response (peak area ratio RZ/RZD6)
versus RZ concentration. Calibration curve standard samples and quality control samples were prepared in replicates (n = 6) for analysis. Precision and accuracy for the
back-calculated concentrations of the calibration points
remained within ±15% of their nominal values. However,
for LLOQ, precision and accuracy were within ±20%.
Selectivity and Specificity
The selectivity of the method was determined by using
blank human plasma samples from six different lots to test
potential interferences of endogenous compounds co-eluted
with RZ and RZD6. The chromatographic peaks of RZ and
RZD6 were identified based on their retention times
and MRM responses. The mean peak area of LOQ for RZ
123
R. Mogili et al.
and RZD6 at corresponding retention time in blank samples should not be more than 20% and 5%, respectively.
Limits of Quantification (LOQ) and Detection (LOD)
LOQ was estimated in accordance with the baseline noise
method. The LOQ was estimated at signal-to-noise (S/N)
ratio of 5. The LOQ was experimentally determined using
six injections of RZ at LOQ concentration (Fig. 3b). Signal-to-noise (S/N) ratio was calculated by selecting the
noise region as close as possible to the signal peak that was
at least eight times the width of the signal peak at half
height.
Matrix Effect
The matrix effect due to the plasma matrix was used to
evaluate ion suppression/enhancement in the signal when
comparing the absolute response of QC samples with the
reconstitution samples (extracted blank plasma sample
spiked with analyte). Experiments were performed at MQC
levels in triplicate with six different plasma lots with
acceptable precision (% coefficient of variation, CV)
B15%.
Recovery
Recovery of RZ was evaluated by comparing the mean
peak area of six extracted low, medium and high quality
control samples (0.3, 30.0 and 70.0 ng mL-1) with the
Fig. 3 Chromatograms of
a rizatriptan and rizatriptan-d6
in blank human plasma, and
b rizatriptan and rizatriptan-d6
in human plasma spiked with
RZ 0.10 ng mL-1 and RZD6
30.00 ng mL-1 [LOQ]
123
mean peak area of six post-spiked with equal amounts of
quality control samples.
Similarly, recovery of RZD6 was evaluated by comparing the mean peak area of extracted quality control
samples with the RZD6 in post-spiked samples with the
same amount of RZD6.
Stability
LQC and HQC samples (n = 6) were retrieved from a deep
freezer after three freeze–thaw cycles according to clinical
protocol. Samples were stored at -10 to -30 °C in three
cycles of 24, 36 and 48 h. In addition, the long-term stability of RZ in QC samples was also evaluated after
64 days of storage at -10 to -30 °C. Post-spiking stability
was studied following a 107 h storage period in an autosampler tray. Benchtop stability was studied for a 26.5 h
period. Stability samples were processed and extracted
along with freshly spiked calibration curve standards. The
precision and accuracy for the stability samples must be
within 15% and ±15%.
Analysis of Human Samples
The bioanalytical method described above was used to
determine RZ concentrations in plasma following oral
administration to healthy human volunteers. Volunteers
gave informed consent before participation in the study,
and the study protocol was approved by the institutional
ethics committee (IEC) as per Indian Council of Medical
Determination of Rizatriptan in Human Plasma by Liquid Chromatography
Research (ICMR) guidelines. Each of 25 healthy human
volunteers was administered a 10 mg dose (one 10 mg
tablet) by oral administration with 240 mL drinking water.
Maxalt tablet 10 mg (Merck and Co., USA) was used as
the reference product, and rizatriptan tablet 10 mg as the
test product. Blood samples were collected pre dose (0 h,
5 min prior to dosing) followed by further samples at 0.25,
0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, 4, 5.5, 7, 9, 11, 13 and 16 h.
After dosing, 5 mL blood was collected each time in
vacutainers containing K2EDTA. A total of 34 samples (17
time points, test and reference product) were collected by
centrifugation at 3,200g at 10 °C for 10 min and stored
below -30 °C until sample analysis. Test and reference
were administered to the same human volunteers under
fasting conditions separately with a gap of 7 days washing
period as per approved protocol.
Pharmacokinetics and Statistical Analysis
Pharmacokinetics parameters from human plasma samples
were calculated by a non-compartmental statistic model
using WinNon-Lin5.0 software (Pharsight, USA). Blood
samples were taken for a period 3–5 times the terminal
elimination half-life (t1/2). Plasma RZ concentration–time
profiles were visually inspected, and Cmax and Tmax values
were determined. The area under the curve from 0 to t, i.e.
AUC0–t, was obtained using the trapezoidal method.
AUC0–? was calculated up to the last measurable concentration, and extrapolations were obtained using the last
measurable concentration and the terminal elimination rate
constant (Ke). Ke was estimated from the slope of the terminal exponential phase of the plasma of the RZ concentration–time curve using the linear regression method. t1/2
was then calculated as 0.693/Ke. AUC0–t, AUC0–? and
Cmax bioequivalence were assessed using analysis of variance (ANOVA) and standard 90% confidence intervals
(CIs) of the test/reference ratio. Bioequivalence was considered when the ratio of averages of log-transformed data
was within 80–125% for AUC0–t, AUC0–? and Cmax [22–
24].
chromatographic optimization and extraction optimization
were carefully carried out to obtain the best results. The
MS optimization was performed by direct infusion of
solutions of both RZ and RZD6 into the electrospray ionization (ESI) source of the mass spectrometer. Other
parameters, such as the nebulizer and heater gases,
declustering potential (DP), entrance potential (EP) and
collision energy (CE) were optimized to obtain better spray
shape, resulting in better ionization and droplet drying to
form the protonated ionic RZ and RZD6 molecules.
A CAD product ion spectrum for RZ and RZD6 yielded
high-abundance fragment ions of m/z (amu) 201.2 and
m/z (amu) 207.1, respectively (Fig. 2b, d). Chromatographic conditions, especially column selection, mobile
phase composition and nature, were optimized through
several trials to achieve the best resolution and increase the
signal of RZ and RZD6. Separation was tried using various
combinations of mobile phase with a variety of columns
such as YMC Pack pro C18, RP-Amide, Ascentis Express
RP-amide, X-Bridge, Discovery Cyano and Kromasil
100-5CN. After the MRM channels were tuned, the mobile
phase was changed from more aqueous phase to organic
phase to obtain a fast and selective LC method. Good
separation and elution were achieved using 10 mM
ammonium formate:acetonitrile (20:80 v/v) as the mobile
phase, at flow rate of 0.5 mL min-1 and injection volume
of 5 ll. Chromatographic analysis of the analyte and IS
was initiated under isocratic conditions with the aim of
developing a simple separation process with short runtime.
A simple LLE technique was used for extraction of RZ and
RZD6 from plasma samples.
Selectivity and Specificity
Analysis of RZ and RZD6 in MRM mode was highly
selective with no interfering compounds (Fig. 3a). Chromatograms obtained from plasma spiked with RZ
(0.1 ng mL-1) and RZD6 (30.0 ng mL-1) are shown in
Fig. 3b.
Limits of Quantification (LOQ) and Detection (LOD)
Results and Discussion
Method Development and Validation
The goal of this research is to develop and validate a
simple, selective, sensitive, rapid, rugged and reproducible
assay method for quantitative determination of RZ in
plasma samples. To develop a simple and easily applicable
method for RZ assay in human plasma for pharmacokinetic
study, HPLC with MS/MS detection was selected as the
method of choice. Mass parameter optimization,
The LOQ and LOD for this method were found to be
0.1 ng mL-1 and 12.5 fg with S/N above 12 at the LOQ
and above 3 at the LOD.
Linearity, Precision and Accuracy
Calibration curves were plotted as instrument response
(peak area ratio RZ/RZD6) versus RZ concentration. Calibration was found to be linear over the concentration range
of 0.1–100.0 ng mL-1. The relative standard deviation
123
R. Mogili et al.
(RSD) was less than 3.2%, and the accuracy ranged from
97.1% to 103.0%. The determination coefficients (r2) were
above 0.9981 for all curves. These results indicate adequate
reliability and reproducibility of this method within the
analytical range (Table 1). The precision and accuracy of
this method were determined by calculating the intra- and
inter-batch variations at three concentrations (0.3, 30.0 and
70.0 ng mL-1) of QC samples in six replicates. As shown
Table 1 Details of calibration curves for validation
Spiked plasma
concentration
(ng mL-1)
RSDa (%)
(n = 5),
precision
(%CV)
Concentration
measured
(mean ± SD)
(ng mL-1)
Accuracy
(%)
0.1
0.1 ± 0.0
1.0
99.0
0.2
0.2 ± 0.0
2.5
101.5
1.0
1.0 ± 0.0
2.6
100.1
5.0
4.9 ± 0.1
2.7
98.9
10.0
10.3 ± 0.1
1.8
103.0
20.0
19.9 ± 0.4
2.1
100.0
40.0
40.1 ± 1.2
3.0
100.4
60.0
60.5 ± 1.4
2.4
100.8
80.0
77.7 ± 1.7
2.3
97.1
100.0
99.0 ± 1.6
3.2
99.0
a
[Standard deviation/mean concentration
n = number of replicates for each level
measured] 9 100,
in Table 2, intra- and inter-day precision within 1.7–3.1%
and 2.8–3.7% and accuracy within 96.0–101.7% and
99.7–101.4% were obtained for RZ.
Stability (Freeze–Thaw, Autosampler, Benchtop
and Long Term)
Quantification of RZ in plasma subjected to three freeze–
thaw (-30 °C to room temperature) cycles showed the
stability of RZ. No significant degradation of RZ was
observed even after 107 h storage periods in an autosampler tray, with final concentration of RZ between 95.0%
and 101.0%. In addition, the long-term stability of RZ in
QC samples after 64 days of storage at -30 °C was also
evaluated. The concentrations ranged from 96.0% to
101.0% of the theoretical values. These results confirm the
stability of RZ in human plasma for at least 64 days at
-30 °C. Benchtop stability for 26.5 h was proved for
RZ with concentrations ranging from 96.6% to 99.0%
(Table 3).
Matrix Effect
The CV% of ion suppression/enhancement in the signal
was found to be 3.4% at the MQC level for RZ, indicating
that the matrix effect on ionization of the analyte is within
the acceptable range.
Table 2 Precision and accuracy (analysis of spiked plasma samples at three different concentrations)
Spiked plasma
concentration
(ng mL-1)
Within-run (N = 1)
Between-run (N = 5)
Concentration measured
(n = 6) (ng mL-1)
(mean ± SD)
a
RSD (%) or
precision
(% CV)
Accuracy
(%)
Concentration measured
(n = 30) (ng mL-1)
(mean ± SD)
RSDa (%) or
precision
(% CV)
Accuracy
(%)
0.3
0.2 ± 0.0
3.1
96.0
0.2 ± 0.0
3.7
99.7
30.0
30.5 ± 0.5
1.7
101.7
30.4 ± 0.8
2.8
101.4
70.0
69.8 ± 1.2
1.8
99.8
70.2 ± 2.2
3.1
100.4
a
[Standard deviation/mean concentration measured] 9 100, N = number of runs, n = number of replicates in each run
Table 3 Stability of rizatriptan in human plasma samples
Spiked plasma
concentration
(ng mL-1)
Room-temperature stability
Processed sample stability
Long-term stability
26.5 h
107.0 h
64 days
Concentration
measured (n = 6)
(ng mL-1)
(mean ± SD)
a
RSD
(n = 6)
(%)
Concentration
measured (n = 6)
(ng mL-1)
(mean ± SD)
a
RSD
(n = 6)
(%)
Concentration
measured (n = 6)
(ng mL-1)
(mean ± SD)
Freeze–thaw stability
Cycle 3 (48 h)
a
RSD
(n = 6)
(%)
Concentration
measured (n = 6)
(ng mL-1)
(mean ± SD)
RSDa
(n = 6)
(%)
0.3
0.2 ± 0.0
1.3
0.2 ± 0.0
2.6
0.2 ± 0.0
2.0
0.2 ± 0.0
0.8
70.0
69.3 ± 1.8
1.6
71.1 ± 2.7
3.9
68.8 ± 1.2
1.5
69.3 ± 0.5
1.3
a
[Standard deviation/mean concentration measured] 9 100
123
Determination of Rizatriptan in Human Plasma by Liquid Chromatography
Recovery
The extraction recoveries of RZ determined at three different concentrations (0.3, 30.0 and 70.0 ng mL-1) were
found to be 96.8 ± 7.6%, 86.4 ± 5.0% and 93.1 ± 2.2%,
respectively. The overall average recoveries of RZ and
RZD6 were found to be 92.1 ± 6.7% and 91.7 ± 4.5%,
respectively.
Application to Biological Samples
The above validated method was used for determination of
RZ in plasma samples to establish the bioequivalence of a
single 10 mg dose (one 10 mg tablet) in 25 healthy volunteers. Typical plasma concentration versus time profiles
are shown in Fig. 4. The pharmacokinetic parameters of
RZ determined in this study were similar in terms of Cmax,
AUC0–t and AUC0–? to those reported previously [10, 11,
16], even though in our study Tmax was observed at 0.75 h,
the number of human volunteers participating was 25 and
blood sample collection time points 0–16 were different
from other studies [10, 11, 15, 16]. All the plasma concentrations of RZ were in the standard curve region and
remained above 0.1 ng mL-1 (LOQ) for the entire sampling period. Pharmacokinetic details are reported in
Tables 4 and 5.
Conclusions
The proposed method offers significant advantages over
those previously reported in terms of simplicity, sensitivity,
selectivity, recovery, ruggedness and reproducibility. The
major advantage of this method is the use of minimum
volume (0.1 mL) of plasma sample, which greatly facilitates blood sample collection. No side-effects were
observed by the participating volunteers during or after the
Fig. 4 Mean plasma concentrations of test versus reference after a
10 mg dose (one 10 mg tablet) in 25 healthy volunteers
Table 4 Mean pharmacokinetic parameters of rizatriptan in 25
healthy human volunteers after oral administration of 10 mg test and
reference products
Rizatriptan pharmacokinetic details
Pharmacokinetic
parameter
Test
Reference
Mean ± SD
CV (%)
Mean ± SD
CV (%)
Cmax (ng mL-1)
21.5 ± 1.1
8.1
20.4 ± 1.1
8.2
AUC0–t (ng h mL-1)
93.3 ± 2.5
3.7
98.7 ± 3.1
4.2
AUC0–µ (ng h mL-1)
94.4 ± 1.5
3.9
99.7 ± 2.1
4.7
Tmax (h)
0.7
–
1.0
–
t1,2
2.4
–
2.2
–
AUC0–µ area under the curve extrapolated to infinity, AUC0–t area under
the curve up to the last sampling time, Cmax maximum plasma concentration Tmax time to reach peak concentration
Table 5 Pharmacokinetic parameters (test/reference) of rizatriptan
after oral administration of 10 mg of test and reference products in
25 healthy human volunteers
Pharmacokinetic
parameters
Cmax (test/
reference)
AUC0–t (test/
reference)
AUC0–6 (test/
reference)
Test/reference
105.3
94.5
94.68
study. This method was successfully applied in a bioequivalence study to evaluate plasma concentrations of RZ
in healthy human volunteers. It can be concluded that the
two analyzed RZ formulations (reference and test) were
bioequivalent.
Acknowledgments The authors wish to acknowledge support
received from the Indian Institute of Chemical Technology (IICT),
Hyderabad, India for providing literature survey and APL Research
Centre, India for providing clinical samples and research support.
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