Available via license: CC BY-NC-ND 4.0
Content may be subject to copyright.
ORIGINAL ARTICLE
Development and validation of a stability-indicating
LC method for simultaneous determination of
related compounds of guaifenesin, terbutaline
sulfate and ambroxol HCl in cough syrup
formulation
K. Lakshmi Narasimha Rao
a,b,
*
, Ch. Krishnaiah
a
, K. Sudheer Babu
a
,
K. Padmaja Reddy
a
a
Department of Branded Formulations, Analytical Research & Development, Dr. Reddy’s Laboratories, IPDO, Bachupally,
Hyderabad 500 072, AP, India
b
Department of Chemisty, J.N.T. University, Kukatpally, Hyderabad 500 072, AP, India
Received 16 October 2011; accepted 18 January 2012
Available online 28 January 2012
KEYWORDS
Development and validation;
Stability indicating;
HPLC;
Terbutaline sulfate;
Guaifenesin;
Ambroxol HCl;
Impurities;
Syrup formulations
Abstract A new liquid chromatographic method has been developed and validated for the deter-
mination of terbutaline sulfate (TLS), guaifenesin (GFN) and ambroxol HCl (AML), for its poten-
tial impurities in drug substances and drug products. Efficient chromatographic separation was
achieved on X-Terra RP-18 column with a simple mobile phase combination containing a gradient
mixture of solvents A and B at a flow rate of 1.0 mL min
1
and quantitation was carried out using
ultraviolet detection at 222 nm with column temperature of 35 C. The resolution between TLS,
GFN and AML, its associated impurities was found to be greater than 1.5. Regression analysis
shows an r value (correlation coefficient) greater than 0.998. This method was capable to detect
all the process impurities of TLS, GFN and AML, at a level below 0.015% with respect to a test
concentration of 0.125, 5.0 and 1.5 mg mL
1
, respectively. The % RSD for the inter-day and
intra-day precisions for all the impurities of TLS, GFN and AML were found to be less than
3.0. The method has shown good, consistent recoveries. The drugs were subjected to stress condi-
*
Corresponding author at: Department of Branded Formulations,
Analytical Research & Development, Dr. Reddy’s Laboratories, IPDO,
Bachupally, Hyderabad 500 072, AP, India. Tel.: +91 6315699878.
E-mail address: klnarasimha16@yahoo.com (K. Lakshmi Narasimha
Rao).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
Journal of Saudi Chemical Society (2014) 18, 593–600
King Saud University
Journal of Saudi Chemical Society
www.ksu.edu.sa
www.sciencedirect.com
http://dx.doi.org/10.1016/j.jscs.2012.01.006
1319-6103 ª 2012 Production and hosting by Elsevier B.V. on behalf of King Saud University.
Open access under CC BY-NC-ND license.
tions of acid, base, water hydrolysis, oxidation, photolysis and thermal degradation, as prescribed
by international conference on harmonization (ICH).
ª 2012 Production and hosting by Elsevier B.V. on behalf of King Saud University.
1. Introduction
Guaifenesin (GFN/GPN), 3-(2-methoxyphenoxy)-1,2 pro-
panediol, is reported to reduce the viscosity of tenacious spu-
tum and is used as an expectorant (Assimos et al., 1999).
Terbutaline sulfate (TLS/TBL), 1,3-Benzenediol, 5-[2-[(1,1-
dimethylethyl)amino]-1-hydroxyethyl]-, sulfate (2:1) (salt) is a
synthetic b
2
-adrenoceptor that is used as a bronchodilator in
the treatment of bronchial asthma (United States, 32nd
edition, NF27, 2081; Jaber et al., 2002). Ambroxol
hydrochloride (AML), [trans-4-(2-Amino-3,5-dibrombenzyl-
amino)-cyclohexanol hydrochloride] is a mucolytic agent
used in the treatment of respiratory disorders associated
with viscid or excessive mucus. Ambroxol hydrochloride
reduces bronchial hyper-reactivity, stimulates cellular
surfactant production, increases the amount of antibiotic
penetration and thus reduces daily dose of them and exhibits
anti-inflammatory properties as well (Gillissen and Nowak,
1998; Shaikh, 2008). The commonly used cough syrup
ingredients are a cough suppressant mean, an expectorant, a
preservative and as part of the excipients sweeteners,
acidulants, antioxidants, natural or artificial coloring and
flavoring agents. These compounds are contained in the oral
syrup dosage form in different proportions. All these types
of excipient and active ingredients create the critical
selectivity of the analytical method to determine its related
impurities. In the literature, a capillary gas chromatography
(Sharaf and Stiff, 2004) and LC-Tandem mass spectrometry
method (Zhong et al., 2005) has been reported for the
determination of GFN and its combination with
Paracetamol in human plasma, respectively. Determination
of TLS by LC in pharmaceutical preparations using micro-
emulsion as eluent (Assi et al., 2011) and HPLC method
(Kyeong Ho Kim et al., 2001; Johnson and Herring, 2000)
has been reported for the determination of TLS in human
urine and plasma. Simultaneous determination of AML with
other combination drugs (Krishna Veni Nagappan et al.,
2008; Barbas and Heina
¨
nen, 2001) and in-presence of
different preservatives (Koundourellis et al., 2000) in
pharmaceutical preparations has been published. A validated
LC method has been reported for these each individual
actives with other combinational drugs for quantification in
pharmaceutical forms (Steven et al., 2007; Barbas and Galli,
2004; Grosa et al., 2006; Korany et al., 2011; Vasudevan
et al., 2000; Stewart and Wilcox, 2000; Lau and Mok, 1995;
Stavchansky et al., 1995; ICH Harmonised Tripartite
Guideline, 2005). There were no LC methods have been
reported on the combination of these three drugs for the
determination of its related substances in a single method.
Hence, developed a stability-indicating LC method that can
separate and determine the imp-A of TLS, imp-(A,B,C,D) of
GFN, imp-(A,B,D,E) of AML in this specific syrup
formulations. Structures of these related substances and their
chemical names are provided in Fig. 1.
2. Experimental
2.1. Chemicals and reagents
The purity of all chemicals was above 98%. Standards of TLS
(98.7%) and its imp-A (99.5%), GFN (98.7%) and its imp-A
(99.5%), imp-B (99.2%), imp-C (96.0%) and imp-D (99.0%),
AML (98.9%) and its imp-A (100.0%), imp-B (98.8%), imp-
D (99.0%) and imp-E (99.0%) were supplied by Dr. Reddy’s
laboratories limited, Hyderabad, India. HPLC-grade Metha-
nol, Acetonitrile, Ammonium dihydrogen phosphate
(NH
4
H
2
PO
4
) and 1-Heptane sulfonic acid sodium salt chemi-
cals of analytical reagent grade were procured from Merck
(Darmstadt, Germany). High purity water was prepared by
using Milli Q plus water purification system (Millipore, Mil-
ford, MA, USA). X-Terra RP-18 column was procured from
Waters Associates Inc.
2.2. Equipment
Chromatography was performed with Waters Alliance HPLC
system (Milford, USA) that consists of quaternary pump
equipped with a 2695 separation module with inbuilt auto
injector and 2996 photodiode array detector. The output sig-
nal was monitored and processed using Empower-2 software.
Cintex digital water bath was used for hydrolysis studies. The
pH of the solutions was measured by a pH meter (Thermo
Orion model 420A, USA). All solutions were degassed by
ultra sonication (Power sonic 420, Labtech, Korea) and fil-
tered through a 0.45 lm Nylon 66 filter (PALL Life sciences,
USA).
2.3. Chromatographic conditions
The method was developed using Waters X-Terra RP-18,
250 mm length, i.d.-4.6 mm, 5 lm particle size column with
mobile phase containing a gradient mixture of solvents A
and B. Solvent A consists of 0.02 M ammonium dihydrogen
orthophosphate with 1.0% of 1-heptane sulphonic acid
sodium salt buffer, pH adjusted to 2.6 with diluted ortho
phosphoric acid, used as a buffer and mixed with acetonitrile
and methanol in the ratio (950:40:10) (v/v/v). Solvent B con-
sists of 0.02 M ammonium dihydrogen orthophosphate with
1.0% of 1-heptane sulphonic acid sodium salt buffer, pH
adjusted to 9.5 with diluted ammonia, used as a buffer and
mixed with acetonitrile in the ratio (400:600) (v/v). The LC
gradient program (T/%B) was set as 0.01/0, 50/0, 55/25,
100/50, 110/65, 120/80, 121/0, 130/0. The flow rate of the
mobile phase was 1.0 mL min
1
. The column oven tempera-
ture was maintained at 35 C and the wavelength was moni-
tored at 222 nm. The injection volume was 20 lL. The diluent
used as water.
594 K. Lakshmi Narasimha Rao et al.
Open access under CC BY-NC-ND license.
erutcurtSsecnatsbusdetaleR
trans-4-[(2-Amino-3,5-dibromobenzyl)amino]cyclohexanol hydrochloride (AML),
active ingredient
Ar-CH
2
OH: (2-amino-3,5-dibromophenyl)methanol (AML Imp-A), process impurity
trans-4-(6,8-dibromo-1,4-dihydroquinazolin-3(2H)-yl)cyclohexanol (AML Imp-B),
process impurity
cis-4-[(2-amino-3,5-dibromobenzyl)amino]cyclohexanol (AML Imp-D), process
impurity
Ar-CH=O: 2-amino-3,5-dibromobenzaldehyde (AML Imp-E), process impurity
1,3-Benzenediol, 5-[2-[(1,1-dimethylethyl)amino]-1-hydroxyethyl]-, sulfate (2:1) (salt)
(or)
(±)-
α
-[(tert-Butylamino)methyl]-3,5-dihydroxybenzyl alcohol sulfate (2:1) (salt) (TLS)
3,5-dihydroxybenzoic acid (α -resorcylic acid), (TLS Imp-A), process impurity
1,2-Propanediol, 3-(2-methoxyphenoxy)-, (±)-.
(±)-3-(o-Methoxyphenoxy)-1,2-propanediol (GFN), active ingredient
2-Methoxyphenol (Guaiacol) (GFN Imp-A), process impurity
2-(2-methoxyphenoxy)propane-1,3-diol (B-isomer) (GFN Imp-B), process impurity
1,1-oxybis{3-(2-methoxyphenoxy)propan-2-ol}(Biseter) (GFN Imp-C), process
impurity
1,3-bis(2-methoxyphenoxy)propan-2-ol (GFN Imp-D), process impurity
Figure 1 Chemical names and structures for GFN, TLS and AML, its associated impurities.
Development and validation of a stability-indicating LC method 595
2.4. Preparation of stock and standard solutions
Stock solutions of GFN (5.0 mg mL
1
), TLS (0.125 mg mL
1
)
and AML (1.5 mg mL
1
) were prepared individually by dis-
solving respective amount of drug in diluent. Mixed diluted
standard was prepared accordingly to obtain a concentration
of GFN (25 lgmL
1
), TLS (1.25 lgmL
1
) and AML
(15 lgmL
1
), respectively.
2.5. Sample preparation
Syrup sample equivalent to 6.25 mg of TLS (32 g of syrup) was
accurately weighed
and transferred into 50 ml volumetric flask.
Added 15 ml of diluent and sonicated for 15 min with occa-
sional shaking, made up to the volume with diluent and mixed
well. Sample solution contains TLS: 0.125 mg mL
1
, GFN:
5.0 mg mL
1
and AML: 1.5 mg mL
1
concentration. The
Figure 2 Typical chromatograms, (A) sample chromatogram, (B) Placebo chromatogram, (C) sample spiked chromatogram.
596 K. Lakshmi Narasimha Rao et al.
solution was filtered through a 0.45 lm nylon 66 membrane fil-
ter (Fig. 2A).
2.6. Placebo preparation
Transferred an accurately weighed quantity of placebo syrup
equivalent
to
the amount present in the sample preparation
(about 32 g) into 50 ml volumetric flask. Added 15 ml of
diluent and sonicated for 15 min with occasional shaking,
made up to the volume with diluent and mixed well. The solu-
tion was filtered through a 0.45 lm nylon 66 membrane filter
(Fig. 2B).
2.7. Relative response factor determination
Five different concentrated solutions of each of the nine
known
impurities
were prepared in the diluent and chromato-
graphed. The response factors for each of the nine known
impurities were calculated by dividing their individual peak
area responses by their respective concentrations. The response
factor for TLS, GFN and AML from the standard solution
was similarly calculated. The relative response factors for each
of the nine known impurities were then calculated by dividing
their determined response factors by the respective active drug
response factor. Chromatographic determination was pre-
sented in Table 1.
3. Results and discussion
3.1. Method development
The primary chromatographic buffer was selected from the lit-
erature. The
blend sample prepared by spiking the impurities
of 1.3 lgmL
1
of TLS impurities, 25 lgmL
1
of GFN impu-
rities and 15 lgmL
1
of AML impurities prepared in the dil-
uent. Ammonium acetate with glacial acetic acid and
ammonium formate with sodium 1-hexane sulfonate buffers
chosen from the TLS method references, which is present as
least dosage form among the three active drugs in the sample.
The buffer concentration was very high and in this method the
impurity peaks of TLS Imp-B with TLS Imp-C, GFN Imp-A
with GFN Imp-B are co-eluting and AML, AML Imp-
(A,B,D,E) were not eluting even after 150 min of its isocratic
run. The three actives and their impurities giving good
response at the wavelength of 222 nm apart from 276 nm.
The
X-Terra
column was chosen which will withstand the wide
pH range. The separations of the impurities are divided into
two parts to simplify the elution pattern. One is hydrophilic
group i.e. TLS, TLS Imp-(A,B,C), GFN, GFN Imp-(A,B),
Sorbic acid, Sodium saccharin and Methyl paraben which
are eluting in 60 min. Two is hydrophobic group i.e. AML,
AML Imp-(A,B,D,E), GFN Imp-(C,D) and propyl paraben
which are eluting on increase of organic solvent.
For part-1, initial buffer composition chosen as 0.02 M
ammonium dihydrogen
phosphate of pH range between 2.5
to 7.0 by varying the individual organic modifier like metha-
nol, tetrahydrofuran (THF). But all the above conditions, sep-
aration of impurities was not satisfactory. It was found that
the use of 0.02 M ammonium dihydrogen phosphate buffer
prepared by adjusting the pH to 2.6 with ortho phosphoric
acid mixed with selective proportion of acetonitrile (ACN) is
giving the good peak shapes but, TLS, GFN, TLS Imp-B
peaks merging with sorbic acid and sodium saccharin peaks.
Percentage ion pair concentration of 1-heptane sulfonic acid
sodium salt and % ACN composition is studied on the above
buffer pH to get effective resolution in part-1 molecules. 0.1%
of ion pair concentration in buffer with 5% addition of ACN is
giving the good resolution among the above set of molecules
except TLS Imp-B peak which is merging with GFN. 1% addi-
tion of methanol to the solvent A i.e. buffer: methanol: ACN
(940:10:50) (v/v/v) ratio serves the purpose of separation
between TLS Imp-B and GFN.
In part-2, Solvent-B was optimized by preparing the same
buffer
of
solvent-A adjusting the pH to 7.0 and 9.5 with
ammonia solution mixed with acetonitrile in the ratio of
(400:600) (v/v) individually and optimized the gradient elution
with Solvent-A, respectively. Solvent-B containing pH 9.5 buf-
fer giving fast elution with good resolution and good peak
shape among AML, AML Imp-D, AML-Imp-A, GFN-Imp-
C, AML-Imp-B, GFN-Imp-D, AML-Imp-E peaks with pla-
cebo peak of propyl paraben.
The order of elution of peaks throughout run is TLS imp-
A, GFN imp-B, GFN imp-A, sorbic acid, TLS imp-B, GFN,
sodium saccharin, TLS, TLS imp-C, methyl paraben, AML,
AML imp-D, AML imp-A, propyl paraben, GFN imp-C,
AML imp-B, GFN imp-D, AML imp-E, respectively.
Table 1 Chromatographic performance data.
S. no. Compound RRT
a
RRF
b
Quantified
c
w.r.t
W.r.t. Peak Value W.r.t. Peak Value
1 TLS Imp-A GFN 0.40 TLS 2.90 TLS
2 GFN Imp-A GFN 0.83 GFN 1.29 GFN
3 GFN Imp-B GFN 0.72 GFN 1.00 GFN
4 GFN Imp-C AML 1.19 GFN 0.98 GFN
5 GFN Imp-D AML 1.29 GFN 1.28 GFN
6 AML Imp-A AML 1.12 AML 0.72 AML
7 AML Imp-B AML 1.25 AML 0.27 AML
8 AML Imp-D AML 1.06 AML 0.47 AML
9 AML Imp-E AML 1.41 AML 0.68 AML
a
Relative retention times (RRT) were calculated with respect (w.r.t) to peak.
b
Relative response factor were calculated with respect to peak.
c
%Impurity quantification were calculated with respect to peak.
Development and validation of a stability-indicating LC method 597
In this method, TLS imp-B and TLS imp-C impurities were
not determined due to poor peak shape and less resolution
with GFN and TLS, respectively. This method is not specific
for those two stated impurities. AML imp-C is not related to
process synthesis of API source. AML imp-C and AML
imp-E are geometrical isomers and AML imp-C is not stable,
converts immediately to AML imp-E. Hence, AML imp-C is
not considered as a part of method development.
3.2. Validation of the method
The proposed method was validated as per ICH guidelines.
3.2.1.
Precision
and intermediate precision
Precision of the method verified by repeatability and checked
by
injecting
six individual preparations of cough syrup sample
(TLS: 1.25 mg, GPN: 50 mg and AML: 15 mg present per each
5 ml of syrup) spiked at 0.50% of its GFN Imp-(A, C, D),
1.0% of TLS Imp-A, GFN Imp-B and AML Imp-(A,B,D,E)
with respect to its active drug concentration present in sample.
The intermediate precision of the method was also evaluated.
%RSD for each impurity concentration was calculated and
observed that the results are less than 5.0% for all its known
impurities in precision and intermediate precision studies.
Spiked sample chromatogram presented in (Fig. 2C).
3.2.2. Specificity
Specificity is the ability to assess unequivocally the analyte in
the
presence
of components that may be expected to be present
such as impurities, degradation products and matrix
components.
3.2.2.1. Placebo Interference. Chromatogram of placebo syrup
preparation
and
sample preparation was cross checked and
found that there is no peak interference at the retention times
of TLS, GFN, AML and its respective known impurities. Pla-
cebo and sample peaks are well resolved.
3.2.2.2. Forced degradation. The specificity studies were per-
formed
on
syrup sample as well as individual active drug sub-
stances to provide an indication and identification of the
generated impurities of the respective drug substance. Inten-
tional degradation was attempted to stress condition of acid
(0.1 N HCl at 60 C/30 min), base (0.1 N NaOH at 60 C/
30 min), hydrolytic (60 C/1 h), oxidation (1.0% H
2
O
2
at
40 C/1 h), photolytic (UV/Not less than 200 watt hours-
square meter
1
, VIS/Not less than 1.2 million lux h) and ther-
mal (80 C/8 h) to evaluate the ability of the proposed method
to separate the impurities of GFN, TLS and AML from its
degradation products. Peak purity test was carried out for
the GFN, TLS and AML peaks as well as known impurities
by using PDA detector in stress samples. Degradation was
found in almost all above stress conditions, The assay of
stressed samples was calculated against a qualified reference
standard and the mass balance was found close to 99.1%
(% assay + % sum of all its compounds + % sum of all its
degradants, respectively). Microwave degradations were very
fast and comparable to the conventional way of the refluxing
method (Fig. 3 A-3D).
3.2.3. Accuracy
The LC chromatogram of spiked sample at 0.5% level of three
impurities
of
GFN Imp-(A, C, D), 1.0% level of six impurities
of TBL-Imp A, GFN Imp-B, AML Imp-(A, B, D, E) in cough
syrup sample. Standard addition and recovery experiments
were conducted on real sample to determine accuracy of the
related substance method. The study was carried out in tripli-
cate using five concentration levels from LOQ to 1.9 lgmL
1
for TLS Imp-A, LOQ to 37.0 lgmL
1
for GFN Imp-(A, C,
D), LOQ to 74.0 lgmL
1
for GFN Imp-B and LOQ to
22.3 lgmL
1
for AML Imp-(A, B, D, E). The percentage
recovery of all the impurities in cough syrup samples varied
from 96.2% to 102.9%.
3.2.4. Limits of detection (LOD) and quantification (LOQ)
The LOD and LOQ for GFN, TLS and AML and its impuri-
ties
were
determined at a signal-to-noise ratio of 3:1 and 10:1,
respectively, by injecting a series of dilute solutions with
known concentrations. LOD was obtained at 0.015% level
whereas LOQ was obtained at 0.05% level with respect to its
active drug concentration. Precision and accuracy study were
also carried out at the LOQ level by preparing the six individ-
ual spiked preparations and the results are found to be well
within the limits.
3.2.5. Linearity
Linear calibration plot for the related substance method was
established at
5 determinations over the calibration range
tested, i.e. LOQ (0.05%) to 1.5% for impurities with respect
to its active drug concentration. Linearity test solutions were
prepared by diluting the pure stock solutions of the drug sub-
stance to the required concentrations. The solutions were pre-
pared at five concentration levels from LOQ to 150% of the
specification level (LOQ-0.05%, 0.50, 0.75, 1.0 and 1.50%
for TLS imp-A, GFN imp-B, AML imp(A, B, D, E); LOQ-
0.05%, 0.25, 0.375, 0.50 and 0.75% for GFN Imp (A, C,
D)). The correlation coefficient obtained was greater than
0.998. The peak area versus concentration data was treated
by least-squares linear regression analysis. The results show
that good correlation existed between the peak area and the
concentration of TLS, TLS imp-A, GFN, GFN imp-A,
GFN imp-B, GFN imp-C, GFN imp-D, AML imp-A, AML
imp-B, AML imp-D and AML imp-E, respectively.
3.2.6. Solution stability and mobile phase stability
The stability of TLS, GFN, AML and their impurities in solu-
tion
for
the related substance method was determined by leav-
ing precision spiked sample solution in a tightly capped
volumetric flask and diluted standard solution at room temper-
ature for 48 h and measuring the amounts of the nine impuri-
ties at every 12 h interval. The stability of mobile phase was
also determined by analyzing freshly prepared solutions of
TLS, GFN, AML and their impurities at 12 h intervals for
48 h. The sample, diluted standard solution and mobile phase
are stable up to 24 h at room temperature.
3.2.7. Robustness
In all the deliberative varied chromatographic conditions (flow
rate,
column
temperature and composition of organic solvent),
all analytes were adequately resolved and elution orders
remained unchanged. The USP resolution between critical
598 K. Lakshmi Narasimha Rao et al.
pairs like GFN imp-A, GFN is 1.5 with sorbic acid peak and
tailing factor for all the actives and its impurities was less than
2.0. The variability in the estimation of GFN, TLS and AML
impurities was within ±5%.
Figure 3 Forced degradation chromatograms, (A) sample in 0.1 N HCl condition, (B) sample in 0.1 N NaOH condition, (C) sample in
1% Peroxide condition, (D) sample in water condition.
Development and validation of a stability-indicating LC method 599
4. Conclusion
The proposed HPLC method for simultaneous determination
of related
compounds of GFN, TLS and AML in combined
syrup dosage form is simple, precise, specific and accurate
for analysis could be recorded. It can be employed for the rou-
tine and stability study analysis.
Acknowledgment
The authors are grateful to the Dr. Reddy’s Laboratories Ltd.
(Hyderabad, India)
for providing laboratory facility for this
research work.
References
Assi, K.H., Althanyan, M.S., Clark, B.J., Hanaee, J., 2011. J. Pharm.
Biomed. Anal. 55, 397–402.
Assimos, D.G., Langenstroer, P., Leinbach, R.F., Mandel, N.S., Stern,
J.M., Holmes,
R.P., 1999. J. Endourol. 13, 665.
Barbas, C., Galli, V., 2004. J. Chromatogr. A 1048, 207–211.
Barbas, Coral, Heina
¨
nen, Maarit,
2001. J. Pharm. Biomed. Anal. 24,
1005–1010.
Gillissen, A., Nowak, D., 1998. Respir. Med. 92, 609–623.
Grosa, Giorgio, Del Grosso, Erika, Russo, Roberta, Allegrone,
Gianna, 2006.
J. Pharm. Biomed. Anal. 41, 798–803.
ICH Harmonised Tripartite Guideline (2005) Validation of analytical
procedures: text
and methodology Q2 (R1). United states phar-
macopeia, 29th edn, NF24, 3050.
Jaber, A.M.Y., Daraghmeh, N., Al-Omari, M.M., Sara, Z., Badwan,
A.A., 2002. J. Pharm. Biomed. Anal. 29, 927–937.
Johnson, Julie A., Herring, Vanessa L., 2000. J. Chromatogr. B 741,
307–312.
Korany, Mohamed A., Fahmy, Ossama T., Mahgoub, Hoda, Maher,
Hadir M.,
2011. J. Adv. Res. 2, 121–130.
Koundourellis, John E., Malliou, Eleftheria T., Broussali, Theodora
A., 2000.
J. Pharm. Biomed. Anal. 23, 469–475.
Krishna Veni Nagappan, Meyyanathan SN, Rajinikanth B Raja,
Suresh Reddy,
Jeyaprakash MR, Arunadevi S Birajdar and
Suresh Bhojraj, Research, (2008), J. Pharm. and Tech. 1(4):
Oct–Dec.
Kim, Kyeong Ho, Kim, Hyun Ju, Kim, Jeong-Hwan, Shin, Sang
Deok, 2001.
J. Chromatogr. B 751 (2001), 69–77.
Lau, Oi.-Wah., Mok, Chuen-Shing, 1995. J. Chromatogr. A 693, 45–
54.
Shaikh, K.A., 2008. J. Pharm. Biomed. Anal. 48, 1481–1484.
Sharaf, Maged H.M., Stiff, Dwight D., 2004. J. Pharm. Biomed. Anal.
35,
801–806.
Stavchansky
, Salomon, Demirbas, Sibel, Reyderman, Larisa, Chai,
Chih-Kun,
1995. J. Pharm. Biomed. Anal. 13, 919–925.
Steven II, T.H. Eichhold, McCauley-Myers, David L., Khambea,
Deepa A.,
Thompson, Gary A., 2007. J. Pharm. Biomed. Anal. 43,
586–600.
Stewart, J.T., Wilcox, Meredith L., 2000. J. Pharm. Biomed. Anal. 23,
909–916.
United States pharmacopeia, 32nd edition, NF27, 2081.
Vasudevan, M., Ravisankar, S., Sathiyanarayanan, A., Chandan,
R.S.,
2000.
J. Pharm. Biomed. Anal. 24, 25–31.
Zhong, Dafang, Chen, Xiaoyan, Huang, Jia, Kong, Zhang, 2005. J.
Chromatogr.
B 817, 263–269.
600 K. Lakshmi Narasimha Rao et al.
