A simple and reproducible HPLC method with spectrophotometric detection was developed for determination of rivastigmine in human plasma. Liquid-liquid extraction of rivastigmine and donepezil (as internal standard) from plasma samples was performed with 1-butanol/n-hexane (2:98 v/v) in alkaline condition followed by back-extraction into diluted acetic acid. Chromatography was carried out using a Silica column (250 mm × 4.6 mm, 5 µm) under isocratic elution with acetonitrile-50 mM aqueous sodium dihydrogen phosphate (17: 83 v/v, pH 3.1. Analyses were run at a flow-rate of 1.3 mL/min at of 50°C. The recovery was 90.8% and 95.7% for rivastigmine and the internal standard donepezil, respectively. The precision of the method was 2.6% to 9.1% over the concentration range of 0.5-16 ng/mL for rivastigmine in plasma with a linearity greater than 0.999. The method was specific and sensitive, with a quantification limit of 0.5 ng/mL and a detection limit of 0.2 ng/mL in plasma. The method was used for a bioequivalence study in healthy subjects.
Acute and Subchronic Toxicity?of Teucrium polium Total Extract in Rats
Iranian Journal of Pharmaceutical Research
(2010), 9 (2): 115-121
Received: September 2008
Accepted: March 2009
Copyright ? 2010 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services
High-Performance Liquid Chromatographic
Determination of Rivastig-
mine in Human Plasma for Application in Pharmacokinetic
Hossein Aminia,b* and Abolhassan Ahmadianib
aDepartment of Pharmacology, Faculty of Medicine, Golestan University of Medical
Gorgan, Iran. bDepartment of Pharmacology, Neuroscience Research Center, Saheed
Beheshti University of Medical Sciences and Health Services,Tehran,Iran.
A simple and reproducible HPLC method with spectrophotometric detection was
developed for determination of rivastigmine in human plasma. Liquid-liquid
extraction of rivastigmine and donepezil (as internal standard) from plasma
samples was performed with 1-butanol/n-hexane (2:98 v/v) in alkaline condition
followed by back-extraction into diluted acetic acid. Chromatography was carried
out using a Silica column (250 mm ? 4.6 mm, 5 ?m) under isocratic elution with
acetonitrile-50 mM aqueous sodium dihydrogen phosphate (17: 83 v/v, pH 3.1.
Analyses were run at a flow-rate of 1.3 mL/min at of 50?C. The recovery was
90.8% and 95.7% for rivastigmine and the internal standard donepezil,
respectively. The precision of the method was 2.6% to 9.1% over the
concentration range of 0.5-16 ng/mL for rivastigmine in plasma with a linearity
greater than 0.999. The method was specific and sensitive, with a quantification
limit of 0.5 ng/mL and a detection limit of 0.2 ng/mL in plasma. The method was
used for a bioequivalence study in healthy subjects.
Rivastigmine hydrogen tartrate, i.e.
(−)S-N-ethyl-3-[(1-dimethyl-amino)ethyl]-N-methylphenyl- carbamate hydrogen
tartrate, is a carbamate inhibitor of acetylcholinesterase (AChE) used in the
treatment of mild to moderate Alzheimer?s disease in adults. It is classified as
an intermediate-acting or pseudo-irreversible agent, due to its long inhibition
on AChE of up to 10 h (1). The drug is rapidly (0.8-1.2 h) absorbed orally. Its
elimination half-life is 1-2 h, and it is converted to an inactive metabolite at
the site of action, by passing hepatic metabolic pathways (2). The oral
bioavailability of rivastigmine increases from approximately 35% at 3 mg to
71.7% at 6 mg (3).
Few analytical methods have been reported in the literature for monitoring
plasma levels of rivastigmine with a limit of quantification (LOQ) of 0.2 ng/mL,
which include GC-MS (3-6) and LC-MS (7-10). However, mass spectrometer is
expensive and not readily available in most clinical research laboratories. In
addition, some methods (3-8) require intensive work for sample preparation.
Recently, an HPLC method with fluorimetric detection was also reported (11);
however, its LOQ of 25 ng/mL is not satisfactory.
Rivastigmine has UV absorption at very short wavelengths (12). This very
non-specific UV absorption makes it difficult to develop a specific, selective
and sensitive UV method, particularly for complex media such as plasma. In the
present work, by using a new sample clean-up procedure and chromatographic
separation method, a selective HPLC?UV assay method for rivastigmine in plasma
was developed. The present method was found reliable and applied for a
bioavailability study of rivastigmine capsules in healthy volunteers.
Rivastigmine hydrogen tartrate and donepezil hydrochloride were obtained from
Vasudha Pharma Chemicals Ltd. (Hyderabad, India). HPLC grade methanol and
acetonitrile, and analytical grade n-hexane and 1-butanol were from Merck
(Darmstadt, Germany). All other reagents used were also of analytical grade,
obtained from Merck (Darmstadt, Germany).
The analyses were performed by a Knauer chromatographic system (Berlin, Germany)
equipped with a Smartline 1000 solvent delivery pump, Smartline 2500 ultraviolet
detector (operated at 200 nm), Jet stream column heater and ChromGate
integrator. The samples were injected by a Rheodyne 7725i loop injector with an
effective volume of 100 ?L. A Waters Spherisorb S5 W (250 mm?4.6 i.d.; 5 ?m
particle size) with a Waters Spheisorb S5W guard column (30 mm?4.6 mm i.d.) was
used for the chromatographic separation. The mobile phase comprised of
acetonitrile- 50 mM sodium dihydrogen phosphate (17: 83 v/v), adjusted to pH 3.1
with concentrated phosphoric acid and 4 M sodium hydroxide. Analyses were run at
a flow rate of 1.3 mL/min at 50? C.
The internal standard donepezil hydrochloride was dissolved in methanol to make
concentrations of 0.1 mg/mL. Stock solution of rivastigmine was prepared in
methanol at a free base concentration of 5 mg /50 mL by dissolving 8 mg
rivastigmine hydrogen tartrate in 50 mL of methanol. The stock solutions of the
internal standard and rivastigmine were stored at -20 ?C. Working standard
solutions were prepared daily from stock solutions, by dilution with 0.1% acetic
Calibration curve and quantitation
Seven-point standard calibration curves were obtained by dissolving appropriate
amounts of rivastigmine in plasma samples. The plasma standards ranged from 0.5
to 16 ng/mL. Calibration curves were constructed by plotting peak height ratio
(y) of rivastigmine to the internal standard versus the rivastigmine
concentration (x). A linear regression was used for quantitation. The prepared
calibration standards (1 mL) were pipetted into 4.5 mL polypropylene tubes with
cap (10 ? 70 mm) and stored at -20?C pending analysis.
Extraction was performed by adding 20 ?L of the internal standard (40 ng of
donepezil), 20 ?L of 1 M NaOH and 3 mL of 1-butanol/n-hexane (2:98, v/v) to 1 mL
of plasma in 4.5 mL polypropylene tube and shaking for 2 min. After
centrifugation at 6000 g for 2 min, the whole organic layer was separated and
transferred into another 4.5 mL tube. Then, 100 ?L of 0.1% acetic acid was
added. The mixture was vortex-mixed for 2 min and centrifuged at 6000 g for 2
min. The upper organic phase was discarded completely and a volume of 80 ?L of
aqueous phase was injected into the chromatographic system.
Blank human plasma obtained from 24 healthy volunteers, was assessed as
described above and compared with the standard samples containing rivastigmine
and also the plasma samples from volunteers after administration of rivastigmine,
in order to evaluate the selectivity of the method. The precision and accuracy
of the method were examined by adding known amounts of rivastigmine to pool
plasma (quality control samples). Quality control samples were made from a stock
solution other than that used to prepare the standards, and were not used for
constructing calibration curves. For intra-day precision and accuracy, five
replicate quality control samples at each concentration were assayed on the same
day. The inter-day precision and accuracy were evaluated by the same results
from five different days within the two-weeks period of analyzing the plasma
samples of volunteers. The absolute recoveries (n = 5) were calculated by
comparing the peak heights obtained from the prepared sample extracts with those
found by direct injection of the same concentrations of drug in 0.1% acetic
acid. The LOQ was estimated by analyzing rivastigmine at low concentrations of
the calibration curve. The LOQ was defined as the minimum concentration where
accuracy and precision were still better than 10%. To determine the limit of
detection (LOD), plasma concentrations lower than the minimum end of the
calibration curve were used. The LOD was then defined as the minimum
concentration which caused a signal three times the noise (S/N=3/1).
The stability of rivastigmine was assessed for standard and volunteers? samples
stored at -20?C for up to 3 months. The acceptance margins for all stability
tests were ?10% of the original concentration.
In-vivo pharmacokinetic study
The assay was used for a comparative bioavailability study of two capsule
preparations containing 3 mg rivastigmine. The study protocol was approved by
the Ethics Committee of the Ministry of Health of Iran, and written informed
consent was signed by volunteers.
Twenty four healthy volunteers participated in the study. The study was
conducted using a two-way cross over design, and as a single dose randomized
trial. The two formulations were individually administrated on two different
days separated by a washout period of 7 days, between the two study medications,
to fasted subjects. Food and drinks were not allowed until 3 h after ingestion
of the capsules. Multiple blood samples (5 mL) were collected before and 0.25,
0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6 and 8 h after the dosing. The plasma
was immediately separated by centrifugation and frozen at −20 ◦C until analysis.
A non-compartmental analysis was used in data processing. Maximum plasma
concentration (Cmax) and time to Cmax (tmax) were determined by inspection of
the plasma concentration-time curves. Elimination constant (kel) was determined
by means of least-squares regression of the data from the last 4-6 points of
each plasma concentration-time curve. Plasma half-life (t1/2) was calculated as
ln(2)/kel. AUC0-t, the area under the concentration-time curve from time zero to
the last detectable drug concentration (Ct), was calculated by the linear
trapezoidal rule. The AUC from time zero to infinity (AUC∞) was calculated by
adding AUC0-t to the extrapolated AUC, obtained by dividing Ct by kel.
Results and Discussion
Significant UV absorption for rivastigmine was obtained at wavelengths below 220
nm. Spectrophotometric detection was used at 200 nm, since at this wavelength
rivastigmine gave 1.5, 3 and 6 times higher peaks in comparison to 210, 215 and
220 nm, respectively.
Selection of chromatographic column
Rivastigmine is usually analyzed on a C18 column (7- 9, 11-12). In the present
study, retention of rivastigmine and some other drugs was examined on C18, CN
and silica columns with mobile phases consisting of mixtures of phosphate buffer
and acetonitrile. Interestingly, the results showed that although rivastigmine
and zolpidem (as the other tested drugs) had strong retentions on C18 column,
they had slightly more retentions on silica column in comparison to CN column
(Table 1). These results show that for rivastigmine and zolpidem, in addition to
reversed-phase retention, other retention mechanisms such as hydrogen-binding or
ion-exchange are probably involved. While other drugs and also endogenous plasma
interferences had extremely lower retentions in Silica column in comparison to
C18 or CN column, unusual retention of rivastigmine in Silica column offered
selectivity and more importantly, excellent clear chromatograms. Therefore, a
silica column was selected for rivastigmine assay in plasma.
Neither the efficiency of the Silica column nor the retention of rivastigmine
and the internal standard changed significantly after more than 3000 biological
sample injections. However, the guard column should be replaced after every 200
A mobile phase containing acetonitrile was the first choice at 200 nm, since it
has a lower UV cut-off than methanol. The separation of rivastigmine by varying
mobile phase compositions was investigated. A simple buffered acetonitrile
mobile phase with an acidic pH was found appropriate for the separation.
Addition of triethylamine (a silanol blocking agent) to the mobile phase reduced
sharpness of rivastigmine peak and therefore was avoided as a mobile phase
Higher temperatures increased the column efficiency for rivastigmine and
therefore were used in the present study. Since high temperature may reduce
column life-time, temperatures above 50?C were not tried.
Rivastigmine could be extracted from plasma, using methyl tert-butyl ether (MTBE)
(3, 9). Based on our experiences; MTBE simultaneously extracted a lot of plasma
interferences and was not suitable for HPLC-UV assay of rivastigmine. For the
same reason, ethyl acetate and dichloromethane were also not suitable. A highly
selective extraction of rivastigmine was obtained, using 1-butanol/n-hexane
(2:98 v/v). Higher percentage of 1-butanol in n-hexane was not suitable, since
recovery of rivastigmine was reduced in back-extraction and also plasma
interferences appeared in chromatograms. Evaporation of the extraction solvents
produced interferences in chromatogram and therefore, a back-extraction was
used. While back-extraction into phosphoric acid or perchloric acid produced
incomplete recovery, back-extraction into diluted acetic acid gave reproducible
and high recovery of rivastigmine and the internal standard donepezil. Diluted
acetic acid as back-extraction medium also produced more clear chromatograms
than did phosphoric acid and perchloric acid.
Selection of internal standard
Among drugs tested as internal standard, zolpidem had a similar retention
behavior to rivastigmine in different chromatographic columns. However, its
recoveries was not suitable using the selected extraction procedure. In
contrast, donepezil, citalopram and norverapamil had high recovery in extraction
from plasma using the proposed method. Donepezil was selected as the internal
standard due to its higher retention in silica column, in comparison with
citalopram and norverapamil.
Representative chromatograms of drug-free plasma, plasma containing dissolved
rivastigmine, and plasma samples from volunteers collected after oral dosing
with rivastigmine are shown in Figure 1. The retention times for rivastigmine
and the internal standard were 4.5 and 5.1 min respectively. No interfering
peaks from the endogenous plasma components were observed at the retention time
of rivastigmine or internal standard. In addition, no late-eluting peak was
observed and new samples could be injected every 6 min. Several drugs including
azithromycin, omeprazole, ranitidine, cimetidine ciprofloxacin, ofloxacin,
amoxicillin, cefixime, clavulanic acid, valproic acid, metformin, acyclovir,
diazepam, oxazepam, moclobemide, prazosin, terazosin, loratadine, cyclosporine,
zolpidem, citalopram, sumatriptan, rizatriptan, verapamil and clonazepam were
tested and none of them interfered. The calibration curves were linear over the
concentration range of 0.5?16 ng/mL in human plasma, with a correlation
coefficient greater than 0.999. The limit of quantification was 0.5 ng/mL and
the limit of detection was 0.2 ng/mL. The results of the intra- and inter-day
accuracy and precision determination have been presented in Table 2. The RSDs of
intra-day precision ranged between 2.6 and 6.4%, whereas that of inter-day
precision were between 3.2 and 9.1%. The intra-day mean error between -7.0 and
2.4%, whereas the inter-day mean error was between -5.6 and 2.8%. The mean
absolute recoveries for rivastigmine and internal standard using the present
extraction procedure were 90.8 and 95.7%, respectively. Stability properties of
rivastigmine during storage and freeze-thaw cycles have been reported (7-10). In
the present study, the stability studies demonstrated stability of rivastigmine
in human plasma samples for at least 3 months storage at -20?C.
The proposed method was used for the determination of rivastigmine in plasma
samples bioequivalence study. The plasma rivastigmine profiles for volunteers
after taking two products are shown in Figure 2. The pharmacokinetic parameters
obtained from the two preparations have been summarized in Table 3. The
extrapolated fraction of the AUC0-∞ accounted for only 8-9%, which indicates a
suitability of the analytical method for pharmacokinetic studies.
In conclusion, it could be said that a selective and sensitive HPLC-UV method
for quantification of rivastigmine in human plasma has been developed and
validated. The simple extraction procedure is based on liquid?liquid extraction
followed by back-extraction into diluted acid. The method is time-saving and
cost-effective, and provides the best alternative for mass spectrometry which is
quite expensive and still not simply available in most laboratories. The
sensitivity of the assay is sufficient to follow the pharmacokinetics of
rivastigmine after administration of a low dose of rivastigmine to human
The authors would like to thank Dr. Saberi, the head of Motahari Hospital (Gonbad
Kavous, Iran), for his kind supports. We also thank Mr. Ayyoub Ghezelsefloo and
Mr. Abdolkarim Kazemi for their helps in blood sampling.
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