Journal of Chromatographic Science 2012;50:615– 619

doi:10.1093/chromsci/bms056 Advance Access publication May 2, 2012 Article

An HPLC Method for the Determination of Bisoprolol in Human Plasma

and its Application to a Pharmacokinetic Study
Sevgi Tatar Ulu* and Zeynep Aydoğmuş
Istanbul University, Faculty of Pharmacy, Department of Analytical Chemistry, 34416, Istanbul, Turkey

*Author to whom correspondence should be addressed. Email: [email protected]

Received 22 August 2011; revised 14 October 2011

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A new high-performance liquid chromatographic method is described from Sigma (Steinheim, Germany). Other chemicals were pro-
for the determination of bisoprolol in human plasma. The proposed vided from Merck (Darmstadt, Germany). All solvents were of
method was based on the derivatization of bisoprolol with 4-chloro- analytical grade.
7-nitro-2,1,3-benzoxadiazole in borate buffer at pH 9.5 to yield a fluor-
escent product.
Chromatographic separation of bisoprolol was achieved by using Apparatus and chromatographic conditions
isocratic elution at a flow rate of 1.2 mL/min on a C18 reversed- Fluorescence spectrum of BIS-NBD derivative was recorded by a
phase column (Inertsil, 4 mm, 150 3 4.6 mm) at 4088 C. The mobile RF-1501 Model A Shimadzu (Kyoto, Japan) spectrofluorimeter.
phase used for the analysis was methanol–water (70:30, % v/v). The liquid chromatographic system was Shimadzu Liquid
Fluorescence detector was used at the excitation and emission Chromatography (Kyoto, Japan) consisting of a Model LC 20 AT
wavelengths of 458 and 525 nm, respectively. The method was vali- solvent delivery system with an SIL-20AHT autosampler with a
dated for linearity, limit of detection, limit of quantification, precision, 5-mL loop and a RF-10AXL fluorescence detector. The analytical
accuracy, recovery and system suitability. The assay was linear over column was an Inertsil C18 column (150  4.6 mm i.d., 5 mm)
the concentration range of 10–2000 ng/mL. This method was with a guard column (4  3 mm i.d., Inertsil) packed with the
applied in pharmacokinetic studies of bisoprolol preparations in same material. The mobile phase was comprised of methanol–
healthy volunteers. water (70:30 v/v). The mobile phase was prepared fresh.
Analyses were run at a flow rate of 1.2 mL/min at 408C. The
fluorimetric detector was set at 458 and 525 nm for the excita-
tion and emission wavelengths, respectively.
Introduction
Bisoprolol hemifumarate (BIS) (+)-1-fp-[(2-isopropoxyethoxy)
methyl] phenoxyg-3-isopropyl-amino-2-propanol hemifumarate Preparation of solutions
is indicated for the treatment of hypertension and angina Stock standard solutions of BIS was prepared in methanol at a
pectoris (1, 2). concentration of 1,000 mg/mL and stored at þ48C. These
Several analytical methods have been studied for the deter- were diluted by using methanol to give appropriate working
mination of BIS in plasma and urine samples. Among these (10 mg/mL) solutions.
methods, high-performance liquid chromatography (HPLC) A borate buffer (0.1M) was prepared by dissolving 0.620 g of
(3 –8), liquid chromatography– tandem mass spectrometry boric acid and 0.750 g of potassium chloride in 100 mL of
(LC –MS-MS) (9 –12), liquid chromatography –electrospray water. The pH was adjusted to 9.5 with 0.1M sodium hydroxide
ionization mass spectrometry (LC –ESI-MS) (13–14) and solution and the volume was made up to 200 mL with water.
electrophoresis (15) have been utilized for the determination The NBD-Cl solution was prepared at 2 mg/mL in methanol.
of BIS.
Some methods have also been reported for the determination
of BIS in pharmaceutical preparations. These articles include
Sample preparation and derivatization
HPLC (16–19), voltammetry (20) and spectrophotometry (21).
In this study, a sensitive fluorimetric HPLC method has been Frozen human plasma samples were thawed at ambient tem-
developed. The method is based on the precolumn derivatiza- perature. Plasma samples were extracted employing a liquid –
tion of BIS with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl). liquid extraction method. Calibration curves were constructed
The proposed method was optimized, fully validated and suc- by adding various amounts (10 –2,000 ng) of BIS to aliquots
cessfully applied to the pharmacokinetic study. (1 mL) of drug-free human plasma. To a 1.0-mL aliquot of
plasma samples, 10 mL of IS (50 ng) and 0.2 mL of 1M sodium
hydroxide solution were added. The samples were briefly
mixed and 3 mL of ethyl acetate were added. The mixture was
Experimental vortex-mixed for approximately 1 min. After centrifugation at
Materials 4,500 rpm for 35 min, the organic layer was transferred to
BIS (98%) and ephedrine (99%) as internal standard (IS) were another 5-mL glass tube and evaporated to dryness under a
supplied from Sigma (Steinheim, Germany). Concor 5 mg tablet gentle stream of air at 408C. Onto the residue, 100 mL buffer
was obtained from a local pharmacy. NBD-Cl was purchased and 100 mL NBD-Cl solutions were added and the mixture was
# The Author [2012]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
kept at 808C for 20 min in a water bath. It was cooled and acid- Stability
ified with 100 mL of 1 N HCl. The associated compound was The stability of plasma samples under different conditions was
extracted from plasma twice with 2.5 mL chloroform and the evaluated. The stability in the extraction solvent was determined
organic layer was transferred to a tube. The organic phase was at 48C and room temperature. The stability of the derivative in
dried on anhydrous sodium sulfate. A 4.5-mL aliquot of the mobile phase before injection into the HPLC was also tested.
extract was evaporated under nitrogen at 458C. The residue
was then dissolved in 1 mL of the mobile phase and filtered
through a 0.2-mm membrane filter. Typically, 5 mL aliquots of
Application to pharmacokinetic study
this solution are used for determination by HPLC.
After oral administration of 5 mg of BIS to a healthy 41-year-old
woman volunteer, the blood samples were collected before
Liquid –liquid extraction and at 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10, 12 and 24 h post-dosing.
A liquid –liquid extraction method was chosen. A large group The plasma was rapidly separated by centrifugation and stored

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of extraction solvents such as n-hexane, ethyl acetate, dichloro- at –208C until analysis. The maximum plasma concentration
methane, chloroform and 2-propanol were tested to develop a (Cmax) and the time to reach Cmax (tmax) were the observed
single-step liquid –liquid extraction procedure for a good re- values. The area under the plasma concentration– time curve
covery. Ethyl acetate was found to be a good extracting solvent. (AUC02t) was calculated using the linear trapezoidal rule. The
This was found to be the most optimum condition for sample half-life (t1/2) was calculated as 0.693/ke.
preparation because it resulted in a clean chromatogram.

Results and Discussion

Method validation
Method development and optimization
Validation was carried out following the International
The reactions of BIS and IS with NBD-Cl in borate buffer at pH
Conference on Harmonization (ICH) (22) guidelines determin-
9.5 produce a yellowish fluorescence color. Fluorescence of
ing, linearity, limits of detection, limits of quantification, preci-
BIS-NBD and IS-NBD gave a spectra maximum at lex 458 nm
sion, accuracy, recovery, stability and system suitability.
and lem 525 nm when spectrofluorimetry was utilized
(Figure 1). Additionally, different experimental parameters
affecting the intensity of BIS-NBD derivative were investigated
Linearity
to determine the optimum parameters.
A calibration curve of BIS-NBD derivative was constructed by
The effect of pH on the intensity of the derivative com-
the linear regression using the internal standard technique.
pounds in the range of 8 –10 was examined. The graphic that
The plots of peak area ratios (rPN ¼ PNBIS/PNIS) versus con-
shows the variation of pH on the intensity is in Figure 2.
centrations of the associated compound were employed.
The influence of temperature and its duration on the inten-
sity of the BIS-NBD derivative were also examined. Four differ-
ent temperatures in the range of 50 –808C were investigated
Limit of detection and limit of quantification
for NBD derivation. The best results were obtained at 808C
The limit of detection (LOD) and limit of quantification (LOQ)
within 20 min.
of the drug by the proposed method were determined using
The effect of time and temperature versus intensity of
calibration standards. LOD and LOQ were calculated as 3.3 and
BIS-NBD derivative is shown in Figure 3. We found that it was
10 s/S, respectively, where S is the slope of the calibration
necessary to acidify the reaction mixture to pH 2 (by adding
curve and s is the standard deviation (SD) of intercept of
100 mL 1N HCl) before the measurement was carried out.
regression equation.

Precision and accuracy Chromatographic conditions

The accuracy and precision tests of BIS-NBD derivative were Several parameters were examined for the optimization of
also tested by determining the active compound in plasma at HPLC analysis of the BIS-NBD associated compound. The first
three concentration levels on three different days. The accur- attempt was to find out the consistency of the mobile phase.
acy and precision of the method was expressed by relative Therefore, it was thought that a mobile phase should consist of
mean error (RME) and relative standard deviation (RSD), solvent –double distilled water without any pH adjustment, and
respectively. methanol was preferred as a solvent in this study.
Different mixtures of methanol and water were tried as
mobile phase, including 90:10, 85:15, 80:20, 70:30 and 60:40,
Recovery v/v. The most suitable peaks appeared when a 70:30, v/v
The extraction recovery for plasma at three different concen- solvent system was utilized.
trations of BIS was determined. Known amounts of BIS were The influence of the flow-rate of the mobile phase on the
added to drug-free plasma and the IS was then added. After the peak normalization was then studied. The optimum conditions
derivatization and chromatography processes, the peak areas were defined as: mobile phase consisting of 70:30, v/v metha-
were compared to the peak areas obtained from the aqueous nol–water, flow-rate of 1.2 mL/min and detecting at excitation
solutions of BIS at the same concentration. and emission wavelengths of 458 and 525 nm, respectively.

616 Tatar Ulu and Aydoğmuş

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Figure 1. Reaction between BIS and NBD-Cl.

Figure 2. Effect of pH on the intensity of the reaction of BIS with NBD-Cl.

Figure 3. Effect of time and temperature on the reaction of BIS with NBD-Cl.

Typical retention times were approximately 4.79 min for BIS and
3.46 min for IS (Figure 4). Figure 4. Chromatogram of blank human plasma with NBD-Cl (A); chromatogram of
plasma spiked with 1,000 ng/mL BIS-NBD and 50 ng/mL IS-NBD (B); chromatogram
of plasma sample obtained from a healthy volunteer 3 h after oral administration of
5 mg of BIS (C).

Method validation LOD and LOQ

The LOD and LOQ of BIS in plasma were 3.2 and 10 ng/mL, re-
Linearity spectively. These values are lower than those obtained by many
Calibration plots were constructed by plotting the concentra- other reported methods (16 –20).
tion against BIS-NBD derivative to IS peak area ratio; these
showed good linearity in the 10 –2,000 ng/mL range for Accuracy and precision
plasma. As the data show, the method is much more sensitive Precision and accuracy were tested by spiking three different
than most of the reported methods (16–20). concentrations (10, 500 and 2,000 ng/mL) into the plasma

An HPLC Method for the Determination of Bisoprolol in Human Plasma and its Application to a Pharmacokinetic Study 617
Table I
Intra-Day and Inter-Day Precision and Accuracy of the Assay for BIS (n ¼ 3)

Human plasma
Added concentration Found concentration (ng/mL) Precision Accuracy
(ng/mL) mean + SD (RSD %) (RME %)
Intra-day
10 10.7 + 0.2 1.86 þ7.0
500 497.6 + 1.74 0.35 20.48
2,000 1,899.0 + 1.22 0.64 25.05
Inter-day
10 10.65 + 0.2 1.87 þ6.5
500 497.4 + 1.15 0.23 20.52
2,000 1,898.3 + 1.39 0.07 25.08

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Figure 5. Plasma concentration– time profile of BIS in a healthy volunteer after a
Table II single oral administration of 5 mg.
Recovery results of BIS-NBD from plasma (n ¼ 3)

Concentration added Concentration found (ng/mL) Recovery RSD

(ng/mL) mean + SD (%) (%) Table IV
Pharmacokinetic Parameters for BIS in Healthy Volunteers
Plasma
10 9.76 + 0.19 97.60 1.94 tmax (h) t1/2 (h) Cmax (ng/mL) AUC0 – 24 (h ng/mL)
500 489.0 + 1.73 97.70 0.35
2,000 1,951.8 + 1.21 97.59 0.06 3.0 10.1 31 274.5

Table III Application to a pharmacokinetic study

System Suitability Parameters (n ¼ 3)
After a single oral dose administration of 5 mg of BIS to a
Capacity factor (k’) Tailing factor (T) Theoretical plates (N) HETP healthy volunteer, a maximum plasma concentration of 31 ng/
9.09 1.04 1,4463 69.14 mL (Cmax) was reached at 3 h (tmax). The elimination half-life
of the drug (t1/2) and area under the curve (AUC0 – 24) were
found to be 10.1 h and 274.5 ng x h/mL, respectively (Figure 5,
Table IV). These pharmacokinetic parameter values are similar
samples: these were injected concurrently for three days. The
to reported values (2).
results of the precision and accuracy test of the method in
human plasma are listed in Table I.

Conclusions
Recovery A sensitive fluorescence HPLC method was developed and vali-
The mean recovery results were obtained in the range of 97.59 dated for BIS after precolumn NBD-Cl derivatization in human
to 97.70%. These are highly suitable values for the determin- plasma.
ation of the compounds. Data of the recoveries of BIS are pre- In this study, the purpose of the derivatization reaction is to
sented in Table II. These values are much better than those raise the sensitivity and thus the possibility of working in low
obtained by many other methods (3, 5). concentrations. The advantages of the liquid–liquid extraction
method include good extraction recovery and simple and less
time consuming procedure. In this study, the recovery percentage
Stability of BIS is high (3, 5); the derivatization and extraction processes
NBD derivatives of BIS in the chloroform were stable for at do not take much time. Additionally, according to the other
least four days at 48C and approximately 3 h at room tempera- methods, the retention time is quite short (3, 5). In this study,
ture. The stability in the mobile phase was also tested, and it the purpose of the derivatization reaction is to raise the sensitivity
was found that the samples were stable for at least 3 h at 48C and thus the possibility of working in low concentrations.
and 1.5 h at room temperature (in the autosampler). In summary, this paper describes a sensitive and accurate
HPLC method for the quantitation of BIS. The method is suit-
able to monitor plasma concentrations during clinical pharma-
System suitability cokinetic studies in humans.
To ascertain the resolution and reproducibility of the HPLC
method, system suitability tests were performed using the
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An HPLC Method for the Determination of Bisoprolol in Human Plasma and its Application to a Pharmacokinetic Study 619