Chromatographia (2014) 77:365–371
DOI 10.1007/s10337-013-2606-4
SHORT COMMUNICATION
Stability‑Indicating UPLC Method for the Determination
of Bisoprolol Fumarate and Hydrochlorothiazide:
Application to Dosage Forms and Biological Sample
Sevinc Kurbanoglu · Paula Rodriguez San Miguel ·
Bengi Uslu · Sibel A. Ozkan 
Received: 9 July 2013 / Revised: 11 November 2013 / Accepted: 12 November 2013 / Published online: 4 December 2013
© Springer-Verlag Berlin Heidelberg 2013
Abstract  A sensitive, selective and accurate ultra perfor-
mance liquid chromatographic method has been developed
and validated for the simultaneous determination of biso-
prolol fumarate and hydrochlorothiazide in their combined
dosage forms and as well as in spiked human urine sam-
ples. The separation was achieved on an Acquity UPLC
BEH C18 1.7 μm (2.1 × 50 mm) column, at 40 °C with
mobile phase consisting of acetonitrile:phosphate buffer
(20 mM) at pH 3.0 with a gradient elution at 225 nm. Biso-
prolol fumarate and hydrochlorothiazide were well sepa-
rated in <1.5 min with good resolution and without any
tailing and interference of excipients. The method was fully
validated according to ICH guidelines in terms of accuracy,
precision, linearity and specificity. A linear response was
observed over the concentration range 0.5–150 μg mL−1
for hydrochlorothiazide and 0.5–250 μg mL−1 for biso-
prolol fumarate. Limit of detection and limit of quantita-
tion for hydrochlorothiazide were calculated as 0.01 and
0.03  μg mL−1, respectively, and for bisoprolol fumarate
were 0.07 and 0.21 μg mL−1, respectively. Moreover, biso-
prolol fumarate and hydrochlorothiazide were subjected to
degradation conditions such as hydrolytic, oxidative and
thermal stress conditions to evaluate the ability of the pro-
posed method for the separation of bisoprolol fumarate and
hydrochlorothiazide from their degradation compounds.
S. Kurbanoglu · B. Uslu (*) · S. A. Ozkan 
Department of Analytical Chemistry, Faculty of Pharmacy,
Ankara University, Ankara, Turkey
e-mail: buslu@pharmacy.ankara.edu.tr
P. Rodriguez San Miguel 
Faculty of Pharmacy, Madrid Complutense University,
Madrid, Spain
Keywords  UPLC · Bisoprolol fumarate · UPLC
Hydrochlorothiazide · Urine samples · Pharmaceuticals
Introduction
During the past 30 years, high performance liquid chroma-
tography (HPLC) has proven to be the predominant tech-
nology used in laboratories worldwide. The laboratories
associated with the use of HPLC could not keep pace with
the growing demands for analyzing more samples, faster
and with more reliable results [1–3]. HPLC performance
was limiting their effectiveness. For chromatographic
investigation, researchers used smaller columns, higher
flow rates, higher working temperatures to increase the
diffusion, but then a pressure problem occurred and some
limitations were soon reached [3–5]. A completely new
system designed with advanced technology in the pump,
detector, and auto sampler, etc., development called as
ultra performance liquid chromatography (UPLC), which
involves high performance liquid chromatography. Separa-
tion technique is widely used for the sensitive and selec-
tive determination of pharmaceutical active compounds in
biological samples and their dosage forms [5–8]. UPLC
is a new category of analytical separation science that
retains the practicality and principles of HPLC while cre-
ating a step function improvement in chromatographic
performance [1–3]. Basically, UPLC improves in three
areas, chromatographic resolution, speed and sensitivity of
the analysis, using 2 μm or less size particles and mobile
phases at high linear velocities and instrumentation that
operates at higher pressure with minimal dispersion than
those used in HPLC. Using fine particles reduces solvent
consumption and allows shortening the analysis time. The
combination of these improvements allows maximizing the
13
366
Fig. 1  Molecular structures of a bisoprolol fumarate, b hydrochloro-
thiazide
benefits of this new material and results in narrower peaks
and more concentrated [9–11]. Arterial hypertension is
a chronic disease characterized by a sustained increase in
blood pressure in the arteries. It is associated with morbid-
ity and mortality rates considerably higher, so it is consid-
ered one of the most important problems of public health,
especially in developed countries, affecting nearly a billion
people worldwide [12]. Chronic hypertension is the single
most important modifiable risk for cardiovascular disease.
Combined cardiovascular therapy is necessary [13]. This
therapy usually involves diuretics and beta blockers, such
as those to be treated below, reducing the incidence of
hypertension adverse events [12–14].
Bisoprolol fumarate (BIS), 1-{4-[(2-isopropoxyeth-
oxy)methyl phenoxy}-3-(isopropylamino) propan-2-ol
(Fig. 1a), is a drug belonging to the group of beta blockers,
a class of drugs used primarily in cardiovascular diseases.
It is a highly selective type β1 adrenergic receptor block-
ing agent used for the treatment of hypertension and angina
pectoris. Bisoprolol fumarate is a cardioselective adreno-
ceptor blocking agent without significant membrane stabi-
lizing or intrinsic sympathomimetic activities in its thera-
peutic dose range. At higher doses, bisoprolol fumarate
also inhibits beta 2-adreno-receptors located in bronchial
and vascular musculature. To retain relative selectivity, it is
important to use the lowest effective dose [12–14].
Hydrochlorothiazide (HCT), 6-chloro-1,1-dioxo-3,4-di-
hydro-2H-1,2,4benzothiadiazine-7-sulfonamide (Fig. 1b),
is a diuretic drug of the thiazide class that acts by inhibiting
the kidneys’ ability to retain water. This reduces the vol-
ume of the blood, decreasing blood return to the heart and
thus cardiac output and, by other mechanisms, is believed
to lower peripheral vascular resistance. Moreover, it is a
calcium-sparing diuretic, and hence can help the body get
rid of excess water while still keeping calcium. It is one of
the oldest thiazide diuretics, often described in combina-
tion with other drugs such as β-blockers, ACE inhibitors, or
angiotensin II receptor blockers [14–16].
BIS and HCT have been recently suggested as a
combination therapy for the treatment of hyperten-
sion and chronic heart failure. The widespread use of
these combined compounds and the need for clinical and
13
S. Kurbanoglu et al.
pharmacological studies require fast and sensitive analyti-
cal techniques to assay the drug in pharmaceutical dosage
forms. Reports indicate that low doses of BIS and HCT
demonstrate synergistic and antihypertensive effects and
provide optimal therapeutic benefits in rats [14–16]. The
antihypertensive effects of these agents are additive; hydro-
chlorothiazide significantly increases the antihypertensive
effect of bisoprolol fumarate. BIS and HCT have different
structural and spectral properties, also they have different
polarities and solubilities, spectrophotometric techniques
with limited sensitivities cannot simultaneously measure
the required limits of the quantitation anticipated for both
HCT and BIS. In the literature, simultaneous determination
BIS and HCT was achieved via HPLC methods [17–19].
To the best of our knowledge, there is no stability-indi-
cating UPLC method for the determination of BIS and HCT
with its application to biological samples like human urine.
The aim of the present work firstly is to develop rapid, sen-
sitive, selective, precise and accurate UPLC method for
the simultaneous determination of BIS and HCT in phar-
maceutical dosage forms as well as in spiked human urine
samples. Moreover, this research aimed to perform stabil-
ity tests for the developed UPLC method on BIS and HCT
according to the guidelines.
Experimental
Chemicals and Reagents
HPLC-grade acetonitrile was obtained from Merck (Darm-
stadt, Germany). HPLC-grade water Milli-Q® system
(Millipore, Milford, MA, USA) was used to prepare all
necessary solutions. Hydrogen peroxide and orthophos-
phoric acid were purchased from Sigma Aldrich. Sodium
hydroxide and hydrochloric acid solutions were analyti-
cal grade (Germany). The chromatographic separations
were performed using Acquity UPLC BEH C18 1.7 μm
(2.1 × 50 mm) (Waters, UK) column at 40 °C. It was used
for the method development and validation studies. Other
all chemicals and solvents were analytical reagent grade.
Materials and Methods
The UPLC system consisted of Water Acquity sample man-
ager system, 2996 photodiode array detector, thermostati-
cally controlled column apartment and auto sampler was
used. The control of UPLC system and data processing
were performed using Empower (All Waters, Milford, MA,
USA). pH measurements were carried out using Thermo
Scientific Benchtop pH meter (Orion 3 Star™ Plus,
USA). Ultrasonic heating bath with Ultrasons (J.P., S.A.
Stability-Indicating UPLC Method
Barcelona) was used for all sample preparation steps. For
spiked human urine solution preparation, a NF 200 Centri-
fuge (NUVE, Turkey) was used.
Chromatographic Conditions
Gradient elution was used by changing the flow rate and
mobile phase composition at the same time. Initially
acetonitrile:phosphate buffer composition was set to 15:85
with a flow rate of 0.7 min mL−1. In 0.6 min analysis time
the composition of the mobile phase was changed to 20:80
and within 1.4 min analysis time the flow rate reached
0.9 min mL−1 with a mobile phase composition of 60:40. At
the end of two min, BIS and HCT were eluted from the col-
umn and chromatographic conditions returned to initial con-
ditions of 0.7 min mL−1 flow rate and 15:85 mobile phase.
For all the experiments Acquity UPLC BEH C18 1.7 μm
(2.1  × 50 mm) column was employed as stationary phase
at 40 °C column temperature. In UPLC system, weak and
strong wash solutions were used to wash the system as 85:15
and 50:50 (v/v) water:acetonitrile, respectively. The total run
time was 2 min and injection sample volume, 5 μL.
Preparation of Standard and Spike Human Urine Stock
Solutions
Accurate 10 mg of BIS and HCT were dissolved in metha-
nol, and allowed to fully dissolve by 10-min ultrasonica-
tion in an ultrasonic bath. The required amount of BIS
and HCT were prepared by diluting the stock solutions of
1,000 μg mL−1 BIS and HCT with mobile phase.
Stock samples for spiked human urine samples were
prepared by mixing with 3.6 mL of human urine with
5.4 mL of acetonitrile and 1 mL of 1,000 μg mL−1 BIS and
HCT from their stock solutions. Stock solutions of urine
samples were kept in an ultrasonic bath for 10 min and sub-
sequently another 20 min in centrifugation program with
3,000 rpm. The supernatant was taken and diluted with the
mobile phase to obtain other concentrations of BIS and
HCT in urine. Blank solution was also prepared by mixing
5.4 mL of acetonitrile, 3.6 mL of human urine and 1 mL of
mobile phase. All the other preparation steps were same as
the spiked urine solutions.
Analysis of Pharmaceutical Dosage Forms
Soprano Plus® Film Tablet (Sandoz) was analyzed using
the proposed UPLC method. 10 tablets were accurately
weighed, crushed and finely powdered. An amount of pow-
dered mass equivalent to 10 mg of BIS and 25 mg of HCT
was weighed and transferred into a 10-mL volumetric flask
and then completed to total volume with methanol. To ensure
complete dissolution of the drugs they were ultrasonicated
367
for approximately 10 min. The solutions were filtered and
the filtrate was collected into a clean flask. They were diluted
with mobile phase to achieve final concentration levels of 10
and 25 μg mL−1 HCT BIS in the final solutions.
Validation Studies
The objective of validation of an analytical procedure is to
reveal that it is suitable for its intended purpose. The ana-
lytical procedure refers to the way of performing the analy-
sis including reference standard, generation of calibration
curve, evaluating specificity, accuracy, precision of the pur-
posed method [20–22].
Specificity: Degradation Studies
ICH guidelines recommend performing degradation stud-
ies for establishing stability-indicating nature of the assay
method [23]. Degradation studies consisted of heat in an
oven (at 75 °C), acidic, alkaline hydrolysis and oxidation
of the compounds, to evaluate the ability of the proposed
method to separate bisoprolol fumarate and hydrochlorothi-
azide from its degradation products. Thermal degradation of
BIS and HCT was carried out in their solid state. BIS and
HCT were placed in a controlled-temperature oven at 75 °C
for 1 h. For acidic and oxidative degradation, BIS and HCT
were diluted with 0.1 N HCl and 30 % H2O2 to achieve
100 μg mL−1 BIS and HCT. The solutions were underwent
the described stress conditions for 1 h. Alkaline hydrolysis
of BIS and HCT was conducted using 0.1 N NaOH for 3 h.
Linearity, LOD and LOQ Values
The linearity of the detector response with different con-
centrations of BIS and HCT was evaluated. Stock stand-
ard solutions were prepared by dissolving BIS and HCT in
methanol. Dilution of the standard stock solutions resulted
in standard mixture solutions containing different concen-
trations. The solutions were injected in triplicate into the
UPLC system and calibration graphs were obtained by
plotting area of the peak versus corresponding concentra-
tion. Regression data analysis was performed using least
squares linear regression statistical analysis. The detection
limit of an individual analytical procedure is the lowest
amount of the analyte in a sample which can be detected
but not necessarily quantified as an exact value. The quan-
tification limit of an individual analytical procedure is the
lowest amount of analyte in a sample which can be quan-
titatively determined with suitable precision and accuracy.
Limit of detection (LOD) and limit of quantification (LOQ)
were calculated from the equations of LOD = 3.3 ×  s/m
and LOQ = 10 × s/m [28], using the standard deviation of
response (s) and the slope of the calibration curve (m).
13
368
Precision: Reproducibility Studies
The precision of an analytical procedure expresses the
closeness of agreement between a series of measurements
obtained from multiple sampling of the same homogene-
ous sample. It is usually expressed as the variance, standard
deviation or coefficient of variation of a series of measure-
ments [28]. Precision data were determined by taking five
measurements of peak areas at 10 μg mL−1 on the same
day and three consecutive days for both standards in mobile
phase and spiked human urine samples.
Accuracy: Recovery from Pharmaceutical Dosage Forms
and Spiked Human Urine Samples
The accuracy of the method was evaluated by recovery
studies. By adding 5 μg mL−1 standard solution of BIS
and HCT to pharmaceutical dosage forms containing
10 μg mL−1 BIS and 25 μg mL−1 HCT, the recoveries were
calculated and confirmed by Bias % and RSD % values.
For recovery of BIS and HCT form spiked human urine
solutions, 5 μg mL−1 standard solutions of BIS and HCT
were added to spiked human urine solutions containing
10 μg mL−1 BIS and HCT. Recovery studies were achieved
with RSD % and Bias % values of the results.
Results and Discussions
Method Development of BIS and HCT in UPLC
In order to develop a method within a short analysis time
and reliable peaks that covers system suitability parame-
ters, pH and the composition of the mobile phase, flow rate,
temperature of the column, wavelength, etc., conditions
should be optimized. Whole-spectrum analysis was per-
formed for BIS and HCT. 225 nm was found as optimum
wavelength for the simultaneous assay of both compounds.
Isocratic eluent were tried with different flow rate between
0.3 and 1 min mL−1, hence in order to shorten the method,
gradient eluent was chosen. After trying several mobile
phases with different percentage of acetonitrile, methanol,
different buffers, pH and strength values, the mobile phase
consisting of acetonitrile and phosphate (20 mM H3PO4)
at pH 3.0 was found as optimum. The composition and the
flow rate of mobile phase were changed gradiently. Initially
acetonitrile:phosphate buffer composition was set to 15:85
with a flow rate of 0.7 min mL−1. In 0.6 min analysis time
the composition of the mobile phase was changed to 20:80
and within 1.4 min analysis time the flow rate reached to
0.9 min mL−1 with a mobile phase composition of 60:40.
At the end of 2 min, BIS and HCT were eluted from the
column and chromatographic conditions returned to initial
13
S. Kurbanoglu et al.
Table 1  Results of hydrolytic, oxidizing and thermal hard stress con-
ditions of HCT and BIS
The response Degraded Degraded
stress conditions HCT % BIS %
HCl (0.1 N) 42 6.3
NaOH (0.1 N) 18 19
H2O2 (30 %) 33 19
Heating 75 °C 5 11
Each value is the mean of three experiments
conditions of 0.7 min mL−1 flow rate and 15:85 mobile
phase at 40 °C column temperature.
System Suitability Test
System suitability tests are an integral part of a liquid chro-
matographic method. It is mandatory to perform them dur-
ing validation of the method, in order to improve the method
[24–27]. To make the method suitable, resolution (Rs > 2.0),
tailing factor (T ≤ 2), selectivity factor (α > 1) are the main
system suitability parameters to take into account [24, 25].
In this system, resolution between BIS and HCT was 6.8
and selective factor was calculated as 4.2 between BIS and
HCT. Tailing factor was calculated as 1.3 for BIS and 1.5
for HCT which are acceptable system suitability results.
Validation of the Developed Method
Specificity: Degradation Studies
The specificity of the method for BIS and HCT was evalu-
ated with the degradation products, and stress degradation
studies of the substances can help to identify them. Degra-
dation experiments were designed using acidic hydrolysis,
alkaline hydrolysis, hydrogen peroxide and oven heating.
Obtained results at different times of the unnatural degra-
dation are in Table 1. Figure 2 shows the chromatograms
obtained by the response of the samples under these drastic
conditions. Peak purity test results derived for both BIS and
HCT responses and it is found that responses were success-
fully resolved from degradation products with the proposed
method.
Linearity
The calibration curve was linear in the range of 0.5–
250 μg mL−1 for BIS and 0.5–150 μg mL−1 for HCT with
a correlation coefficient (r) of 0.99 for both compounds.
The interval between the upper and lower concentration
of analyte in the sample demonstrate that the analytical
procedure had a suitable level of precision, accuracy and
Stability-Indicating UPLC Method 369

a b

1
1

2 1 2 2
1
2
2
Detector Response

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2.0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2.0

c 1 d 1

2
2

1 1

2 2

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2.0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2.0

Time (min)

Fig. 2  Degradation of BIS and HCT a in 30 % H2O2 for 1 h, b 0.1 N HCl for 3 h, c at 75 °C for 1 h, d in 0.1 N NaOH for 1 h (1 without degrada-
tion, 2 under degradation conditions)

Table 2  Statistical evaluation Compounds Standard solution Human urine

of the calibration data by UPLC
HCT BIS HCT BIS

Retention time (min) 0.46 1.14 0.46 1.14

Linearity range (μg mL−1) 0.5–150 0.5–250 0.5–10 0.5–30
Slope (mAU μg−1 mL−1) 44,563 9,706.7 49,484 8,926
SE of slope 6.531 × 101 1.195 × 102 2.351 × 102 9.168 × 101
Intercept (mAU) −62,191 −19,291 1,844.7 8,668.9
SE of intercept 4.935 × 104 1.450 × 104 1.200 × 103 1.469 × 103
Correlation coefficient 0.99 0.99 0.99 0.99
LOD (μg mL−1) 0.01 0.07 0.02 0.07
LOQ (μg mL−1) 0.03 0.21 0.06 0.22
Within-day repeatabilitya (RSD %) 0.01 0.01 0.003 0.002
a
  Each value is the mean of five Between-day repeatabilitya (RSD %) 0.01 0.01 0.01 0.001
experiments

linearity. LOD and LOQ values were calculated as 0.07–
0.21 μg mL−1; and 0.01–0.03 μg mL−1 for BIS and HCT,
respectively. Standard error of slope and intercept was also
calculated and the low standard error values of the slopes
and intercepts showed also the precision of the method and
are reported in Table 2.
For spiked human urine studies, the calibration curve
was linear in the range between 0.5 and 10 μg mL−1
for HCT and 0.5–30 μg mL−1 for BIS, with a correla-
tion coefficient (r) of 0.99 for both compounds. LOD
and LOQ values were calculated as 0.07–0.22 and 0.02–
0.06 μg mL−1 for HCT and BIS, respectively, in urine sam-
ples. In Fig. 3 different concentrations of BIS and HCT in
standard and human urine are shown.
Moreover, Soprano Plus® Film Tablet was analyzed
using proposed UPLC method. In Fig. 3a inset, chroma-
togram of Soprano Plus® Film Tablet was shown with
reliable results in the presence of excipients. RSD % and
Bias % values are shown in Table 3 for the analysis of BIS
and HCT in pharmaceutical dosage forms.

13
370 S. Kurbanoglu et al.

Fig.  3  a Different concentra-

tions for standard BIS and
HCT, b BIS and HCT in human
urine 1 10 μg mL−1 BIS and
HCT, 2 25 μg mL−1 BIS and
HCT, 3 50 μg mL−1 BIS and
HCT. Inset Chromatogram of
Soprano Plus® Film Tablet
including 25 μg mL−1 HCT and
10 μg mL−1 BIS

Precision: Repeatability Studies and recovered from the tablet sample with good RSD and
Bias values as shown in Table 3.
The parameter of precision considered was repeatability
(within day and between days), summarized in Table 2.
Based on results of Table 2, there was no significant differ- Conclusions
ence between them for the assay of both standard solutions
of BIS and HCT and spiked human urine samples. A selective, sensitive, precise and accurate UPLC method
for the simultaneous determination of bisoprolol fumarate
Accuracy: Recovery Studies and hydrochlorothiazide has been developed, optimized
and validated.
The accuracy of the proposed method was proved by recov- Once the optimized conditions were achieved, calibration
ery studies. 5 μg mL−1 BIS and HCT were added to phar- studies and the overall capabilities of a number of analyti-
maceutical dosage forms containing 10 μg mL−1 BIS and cal procedures in combination were appreciated in order to
25  μg mL−1 HCT. 5 μg mL−1 BIS and HCT were added demonstrate that the procedure is suitable for its intended
purpose. Also the proposed method can be easily applied to
Table 3  Results of the assay and the recovery analysis of BIS and
the stress degradation studies of BIS. Therefore, taking into
HCT in pharmaceutical dosage forms account the results, the developed method appears to be the
first direct stability-indicating method for the simultaneous
Standard solution Human urine
assay of the studied drugs using UPLC. The proposed UPLC
HCT BIS HCT BIS method is a powerful, convenient and useful technique for
Labeled claim (mg) 25 10 – –
the analysis of drugs. For this reason, the stability-indicating
Amount found (mg)a 25.08 9.98 – –
method was applied to analyzing of BIS and HCT in their
RSD (%) 0.002 0.01 – –
pharmaceutical dosage form and in spiked human urine sam-
ple. The suggested method may be used for the routine anal-
Bias (%) −0.33 0.16 – –
ysis of both drugs in differently equipped laboratories.
Added (mg) 5.00 5.00 5.00 5.00
Founda (mg) 4.97 5.01 5.17 4.87
Recovery (%) 99.72 100.12 103.42 99.54 References
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