Determination of labetalol hydrochloride in drug formulations by spectrophotometry

Article abstract of DOI:10.1002/jccs.200700028

A validated, selective and sensitive spectrophotometric method has been developed for the determination of labetalol hydrochloride in commercial dosage forms. The method is based on the coupling reaction of positive diazonium ion of 4-aminobenzenesulfonic acid with phenolate ion of labetalol to form a colored azo compound which absorbs maximally at 395 nm. Under the optimized experimental conditions, the color is stable up to 2 h and Beer's law is obeyed in the concentration range of 0.8-17.6 μg mL^-1 with a linear regression equation of A = 4.84 × 10^-4 + 7.864 × 10^-2 C and coefficient of correlation, r = 0.9999. The molar absorptivity and Sandell's sensitivity are found to be 2.874 × 10⁴ L mol^-1 cm^-1 and 0.013 μg cm^-2 per 0.001-absorbance unit, respectively. The limits of detection and quantitation of the proposed method are 0.08 and 0.23 μg mL^-1, respectively. The intra-day and inter-day precision variation and accuracy of the proposed method is acceptable with low values of standard analytical error. The recovery results obtained by the proposed method in drug formulations are acceptable with mean percent recovery ± RSD of 99.97 ± 0.52 - 100.03 ± 0.63%. The results of the proposed method compared with those of Bilal's spectrophotometric method indicated excellent agreement with acceptable true bias of all samples within ± 2.0%.

Full text of DOI:10.1002/jccs.200700028

Journal of the Chinese Chemical Society, 2007, 54, 185-196
185
Determination of Labetalol Hydrochloride in Drug Formulations by
Spectrophotometry
Nafisur Rahman,* Habibur Rahman and Syed Najmul Hejaz Azmi
Department of Chemistry, Aligarh Muslim University, Aligarh-202002, Uttar Pradesh, India
A validated, selective and sensitive spectrophotometric method has been developed for the determina-
tion of labetalol hydrochloride in commercial dosage forms. The method is based on the coupling reaction
of positive diazonium ion of 4-aminobenzenesulfonic acid with phenolate ion of labetalol to form a col-
ored azo compound which absorbs maximally at 395 nm. Under the optimized experimental conditions,
the color is stable up to 2 h and Beer’s law is obeyed in the concentration range of 0.8-17.6 mg mL-1 with a
linear regression equation of A = 4.84 × 10-4 + 7.864 × 10-2 C and coefficient of correlation, r = 0.9999.
The molar absorptivity and Sandell’s sensitivity are found to be 2.874 × 104 L mol-1 cm-1 and 0.013 mg cm-2
per 0.001-absorbance unit, respectively. The limits of detection and quantitation of the proposed method
are 0.08 and 0.23 mg mL-1, respectively. The intra-day and inter-day precision variation and accuracy of
the proposed method is acceptable with low values of standard analytical error. The recovery results ob-
tained by the proposed method in drug formulations are acceptable with mean percent recovery RSD of
99.97 0.52 - 100.03 0.63%. The results of the proposed method compared with those of Bilal’s spectro-
photometric method indicated excellent agreement with acceptable true bias of all samples within 2.0%.
Keywords: Labetalol hydrochloride; Azo compound; Validation; Pharmaceutical formulations;
Spectrophotometry.
INTRODUCTION
Labetalol hydrochloride, i.e., Benzamide, 2-hydroxy-
uid chromatographic method, respectively. Several analyt-
ical methods such as thin layer chromatography (HPTLC),5,6
high performance liquid chromatography (HPLC),7,8 liquid
chromatography (LC),9,10 liquid chromatography coupled
with mass spectrometry (LC-MS),11,12 gas chromatography
coupled with mass spectrometry (GC-MS)13 capillary elec-
trophoresis (CE),14 electrochemical methods15 and flouri-
metry16 are reported for the estimation of labetalol hydro-
chloride. The determination in biological fluids normally
requires the use of trace analysis techniques, higher instru-
mentation such as HPLC, LC, CE, cyclic voltammetry,
fluorometry and hyphenated techniques like LC-MS,
GC-MS, and inductively coupled plasma-mass spectrome-
try which normally makes the method much more expen-
sive. In addition to this, these methods require long and te-
dious pretreatment of the samples and laborious clean up
procedures prior to analysis. UV-visible spectrophotome-
try is the technique of choice even today in the laborato-
ries of research, hospitals and pharmaceutical industries
due to its low cost and inherent simplicity. The analytical
procedures based on spectrophotometry17-22 have been fre-
5-[1-hydroxy-2-[(1-methyl-3-phenylpropyl)amino]ethyl]-,
monohydrochloride (CAS No. 32780-64-6, M.W. 364.87)
is an adrenergic b-receptor blocking agent used in the treat-
ment of hypertension, which exhibits both a and b-adreno-
ceptor blocking activity, and because of its use as a doping
agent in sports, this drug has been added to the list of for-
bidden substances issued by the International Olympic
Committee.1 The drug is used to induce hypotension during
surgery as it decreases blood pressure more rapidly than
other beta blockers. The drug is quite sensitive, even a
small dose of the drug gives sufficient blockage, thus indi-
cating that the drug is very much confined to the cardio pro-
tective effects. Hence, it is a must to quantitate the amount
of labetalol hydrochloride in drug formulations. The drug
is official in Martindale: The Extra Pharmacopoeia.2 The
assay procedure is cited in the monographs of the British
Pharmacopoeia3 and the United States Pharmacopoeia,4
which describe a potentiometric titration method and a liq-
* Corresponding author. E-mail: cht17nr@yahoo.co.in

186 J. Chin. Chem. Soc., Vol. 54, No. 1, 2007
Rahman et al.
EXPERIMENTAL
quently published in reputed journals. Moreover, spectro-
photometry is a well known technique which has been uti-
lized for the determination of isoniazid based on the reac-
tion of drug with 1,2-naphthoquinone-4-sulfonic acid so-
dium in alkaline medium (pH 13).23 Some spectrophoto-
metric methods have been developed for the determination
of labetalol hydrochloride in bulk drug and pharmaceutical
formulations. The quantification of the drug was done
based on the reaction of labetalol hydrochloride with Folin
Ciocalteau’s reagent, 4-aminophenazone, 2,6-dichloroqui-
none chlorimide and ferric alum;24 benzocaine and p-nitro-
aniline in the presence of triethylamine and sodium carbon-
ate, respectively;25 p-N,N-dimethylphenylenediamine and
3-methyl 2-benzothiazolinone hydrazone hydrochloride in
the presence of sodium hypochlorite and ceric ammonium
sulfate to form an intensely colored product that absorbs
maximally at 685 and 545 nm, respectively.26 The quantita-
tion of drug was done in the visible region based on ion-pair
formation between drug and reagents like suprachen violet
3 B26 and wool fast blue BL.27 It is better to avoid develop-
ing the assay procedure using extractive methods to mini-
mize the use of organic solvents such as chloroform and di-
chloromethane, which cause harmful effects on human life.
There is also a possibility of the loss of analyte and other
handling problems in developing extractive spectrophoto-
metric methods. Labetalol hydrochloride is present in rela-
tively large amounts (100 mg per tablet or 50-200 mg per
capsule) in pharmaceutical formulations and the sample
matrix of drug is usually less complex. Therefore, it is im-
portant to develop a spectrophotometric method that avoids
the use organic solvents to determine the amount of drug in
pharmaceutical formulations.
Apparatus and Reagents
The absorbance measurements were made on a Shimadzu
UV-visible 1601 spectrophotometer (Kyoto, Tokyo, Japan)
with matched quartz cells.
All chemicals and reagents were of analytical grade.
Aqueous solutions of 4-aminobenzenesulfonic acid (0.029
M; Fluka Chemie AG, Switzerland), NaNO2 (0.29 M; Otto
Chemie, Mumbai, India) and sodium carbonate (0.28 M;
GR grade, Merck Limited, Mumbai, India) were freshly
prepared. A 0.2 M HCl was also prepared by diluting 1.77
mL of pure HCl (35%, GR grade, Merck Limited, Mumbai,
India) in 100 mL distilled water.
Drug standard and commercial dosage forms
Labetalol hydrochloride as a reference standard was
obtained from Sigma Chemical Company (St. Louis, MO,
USA). A standard solution of labetalol hydrochloride
(0.04%) was prepared in distilled water. Commercial dos-
age forms of labetalol hydrochloride such as Lobet-100 mg
tablets (Samarth Pharma Pvt. Ltd., Mumbai, India) and
Normadate-50 mg capsules (Glaxo Smithkline, Mumbai,
India), were obtained from a local drug store.
Spectrophotometric procedure for the determination
of labetalol hydrochloride
Into a series of 25 mL standard volumetric flasks, 1.5
mL of 0.029 M 4-aminobenzenesulfonic acid, 1.5 mL of
0.29 M NaNO2 and 2.0 mL of 0.2 M HCl were pipetted and
kept in an ice-bath (0-3 °C) for 10.0 min to complete di-
azotization reaction. To each flask, an aliquot of 0.04%
labetalol hydrochloride reference standard solution (0.05-
1.1 mL) was added followed by 4.0 mL of 0.28 M sodium
carbonate and then diluted to volume with distilled water at
room temperature (25 1 °C). The contents of each flask
were mixed well and the absorbance was measured at 395
nm against the reagent blank prepared similarly except for
the drug. The color was stable up to 2 h. The amount of the
drug was computed either from the calibration graph or the
regression equation.
The present paper describes a selective and sensitive
spectrophotometric method for the determination of labe-
talol in drug formulations. The method is based on the reac-
tion of 4-aminobenzenesulfonic acid with dilute HCl and
NaNO2 in a temperature range of 0-3 °C to form an electro-
phile, i.e, positive diazonium ion, which immediately cou-
ples at ortho to the hydroxyl group of the benzene ring of
labetalol in Na2CO3 medium forming a yellow azo product
having lmax at 395 nm. The proposed method is carefully
optimized and validated as per the International Confer-
ence on Harmonisation (ICH) guidelines28 to enhance the
sensitivity, specificity/selectivity and applicability of the
proposed method.
Procedure for the determination of labetalol hydro-
chloride in drug formulations
The powdered contents of 2 capsules of 50 mg
strength (or 1 tablet of 100 mg strength) were taken in 50

Determination of Labetalol Hydrochloride
J. Chin. Chem. Soc., Vol. 54, No. 1, 2007 187
mL distilled water and left for 10 min for complete disper-
sion and then filtered through Whatman No. 42 filter paper
(Whatman International Limited, Kent, UK) in a 250 mL
standard volumetric flask. The residue was washed well
with distilled water for complete recovery of the drug and
then diluted up to the mark with distilled water to give a
concentration of 0.4 mg mL-1. The assay was completed
following the recommended spectrophotometric procedure
for the determination of labetalol hydrochloride.
Scheme I Oxidative degradation of labetalol through
N-dealkylation: I. 2-hydroxy-5-formyl-
benzamide and II. 1-methyl-3-phenylpro-
pylamine
CONH2
OH
CH3
N
H
OH
sodium metaperiodate
in Na2CO3 medium
Preparation of degraded labetalol
Labetalol hydrochloride was oxidized by treating 100
mg of the drug in 10 mL distilled water with 0.5 mL satu-
rated sodium carbonate solution and 2.5 mL of 0.3 M so-
CONH2
OH
CH3
NH2
O
C
dium metaperiodate for 10 min at room temperature (25
1
H
°C). The excess sodium carbonate was neutralized with the
corresponding volume of HCl and then the products were
isolated. The hydrolyzed solution was treated with methyl
isobutyl ketone. Two layers were formed and the organic
layer was separated out. The extract was then evaporated to
dryness at room temperature, and the residue as compound
1 (2-hydroxy-5-formylbenzamide) was crystallized from
distilled water. Compound 2 (1-methyl-3-phenylpropyl-
amine), which was in the aqueous layer, was extracted with
dichloromethane. The extract was evaporated to dryness
and the residue was crystallized in ether. Thin layer chro-
matography was applied using a silica gel G plate and a
mixture of n-propanol-1% ammonia (4:1, v/v) as the mo-
bile phase. The bands corresponding to labetalol, com-
pound 1 (2-hydroxy-5-formylbenzamide) and compound 2
(1-methyl-3-phenylpropylamine) were located under UV
lamp at 254 nm, and the Rf values were found to be 0.70,
0.80 and 0.35, respectively. The spots of compounds 1 and
2 were also detected by spraying with 2,4-dinitrophenyl-
hydrazine in 2M HCl and nynhydrin to give yellow and
purple spots, respectively, thus indicating the presence of
the carbonyl group in compound 1 and the amine group in
compound 2. The literature has confirmed29 that the two
major products of labetalol are 2-hydroxy-5-formylbenz-
amide (I, compound 1) and 1-methyl-3-phenylpropylamine
(II, compound 2) (Scheme I) thus confirming the above
finding.
II
I
Specificity
The sample solutions of labetalol tablets and capsules
were subjected to stress conditions of light, heat, acid, base
and oxidants. All stressed samples were analyzed for labe-
talol content and compared to an unstressed time zero refer-
ence solution. The reference assay value for the unstressed
product was evaluated, and the content of degradation in
the stressed and control samples was calculated relative to
this assay value.
Linearity
The linearity was evaluated by determining labetalol
at ten concentration levels: 0.8, 1.6, 3.2, 4.8, 6.4, 8.0, 11.2,
14.4, 16.0 and 17.6 mg mL-1. Each concentration was ana-
lyzed five times.
Accuracy and precision
Three concentrations within the linearity range were
selected: 1.6, 8.0, and 16.0 mg mL-1 for evaluating accuracy
and precision. Five independent analyses at each concen-
tration level were performed within one day (intra day as-
say) and repeated for five consecutive days (inter day as-
say).
Validation
Recovery studies
The proposed method has been validated for specific-
ity, selectivity, linearity, precision, accuracy, and recovery.
To study the accuracy of the proposed method and to
check the interference from excipients used in the formula-

188 J. Chin. Chem. Soc., Vol. 54, No. 1, 2007
Rahman et al.
tions, recovery experiments were carried out by the stan-
dard addition method. For this, 1.2 mL (or 2.0 mL) of sam-
ple solution (0.4 mg mL-1) was transferred into a 100.0 mL
standard volumetric flask followed by 0.8 mL (or 2.0 mL)
of reference standard solution (0.4 mg mL-1) and volume
was completed to the mark with distilled water. The nomi-
nal value was determined by the recommended procedure.
form a yellow azo compound, which absorbs maximally at
395 nm. In this case the coupling of diazonium ion pro-
ceeds ortho to the hydroxyl group of the benzene ring of
labetalol because the para position in the drug is already oc-
cupied. The reaction sequence is shown in Scheme II.
Optimization of variables
The different parameters responsible for the forma-
tion of diazocoupling colored product were extensively
studied. The optimum conditions for the proposed method
were established by conducting a series of experiments.
RESULTS AND DISCUSSION
Spectral studies
The aqueous solutions of pure labetalol and 4-amino-
benzenesulfonic acid (sulfanilic acid) exhibit absorption
maxima at 208 and 249 nm, respectively. The addition of
NaNO2 and HCl to 4-aminobenzenesulfonic acid causes
diazotization and the resulting product absorbs maximally
at 270 nm. The blank solution is prepared similarly on addi-
tion of sodium carbonate to this diazotized solution, which
causes no shift in the absorption maximum. The blank solu-
tion when added to the drug solution causes a remarkable
shift in the absorption maximum due to the formation of
coupling product, which exhibits an absorption band peak-
ing at 395 nm (Fig. 1). This reaction is exploited to develop
a spectrophotometric method for the determination of
labetalol hydrochloride in drug formulations by measuring
the concrete absorbance at 395 nm.
Effect of time
To optimize the reaction time for color development,
1.74 × 10-3 M 4-aminobenzenesulfonic acid was treated
with 1.74 × 10-2 M NaNO2 and 1.60 × 10-2 M HCl in a 25
Mechanism of the color reaction
It has been reported that primary aromatic amines
such as 4-nitroaniline and 4-aminobenzenesulfonic acid on
treatment with HCl and NaNO2 in a temperature range of
0-3 °C, yield diazonium salt.30 However, only strongly
nucleophilic electron-rich aromatic or heteroaromatic com-
pounds such as phenol can be coupled with diazonium salts
to yield colored azo compounds.31 The diazocoupling reac-
tion can be considered as a proton eliminating condensa-
tion of a positive diazonium ion with another compound
possessing an active hydrogen atom. The substitution of
electrophile usually proceeds para to the active group or
ortho if the para position is already substituted. In a similar
manner, 4-aminobenzenesulfonic acid was diazotized to
form a positive diazonium ion. Labetalol is a water-soluble
antiadrenergic $-blocker possessing an -OH group at-
tached to the benzene ring (phenol) and forms phenolate
ion in sodium carbonate medium.32 The phenolate ion un-
dergoes coupling reaction with positive diazonium ion to
Fig. 1. Absorption spectra of (a) pure drug solution:
10.0 mg mL-1 of labetalol hydrochloride in dis-
tilled water; (b) 8.67 × 10-5 M 4-aminobenzene-
sulfonic acid in distilled water; (c) blank solu-
tion: diazotized solution (2 mL of 1.73 × 10-4 M
4-aminobenzenesulfonic acid + 1.5 mL of 0.29
M NaNO2 + 1.8 mL of 0.2 M HCl ) with 4.0 mL
of 0.28 M Na2CO3 in 25 mL distilled water and
(d) sample solution: 440.0 mg labetalol hydro-
chloride and 4.0 mL of 0.28 M Na2CO3 with
diazotized solution (1.5 mL of 0.029 M 4-ami-
nobenzenesulfonic acid + 1.5 mL of 0.29 M
NaNO2 + 1.8 mL of 0.2 M HCl) in 25 mL dis-
tilled water.

Determination of Labetalol Hydrochloride
J. Chin. Chem. Soc., Vol. 54, No. 1, 2007 189
Scheme II Azo compound of labetalol (yellow colored) formed due to union between the positive diazonium ion
and a phenolate anion in sodium carbonate medium
CONH2
OH
HO3S
NH2
CH3
N
4-aminobenzenesulfonic acid
H
OH
NaNO2 + 2HCl
0 - 3o C
labetalol
Na2CO3 in H2O
_
+
N
HO3S
NCl
CONH2
-
+
NaCl
+
2H2O
O
CH3
N
H
OH
+
N
HO3S
N
phenolate anion
positive diazonium ion
diazo coupling
CONH2
OH
CH3
N
N
SO3H
N
H
OH
Azo coupling product (colored)
mL standard volumetric flask. The content of the mixture
was maintained in an ice bath (0-3 °C) for varied times of
2-14 min and then the flask was taken out. To each flask,
1.0 mL of 0.04% labetalol hydrochloride and 4.48 × 10-2 M
Na2CO3 were added and diluted to volume with distilled
water at room temperature (25 1 °C). The maximum in-
tensity of color was obtained at 7 min of cooling and re-
mained constant up to 14 min. Therefore, 10 min of reac-
tion time was used throughout the determination process.
metric flask and was cooled in an ice bath for 10 min. After
cooling, 400.0 mg labetalol hydrochloride and 4.48 × 10-2
M Na2CO3 were added and diluted to volume with distilled
water at room temperature (25 1 °C). It was observed that
the absorbance measured at 395 nm increased with increas-
ing concentration of 4-aminobenzenesulfonic acid and be-
came constant at 1.16 × 10-3 M; above this concentration
absorbance remained constant (Fig. 2a). Therefore, the
concentration of 1.74 × 10-3 M 4-aminobenzenesulfonic
acid proved to be sufficient for the maximum concentration
of labetalol hydrochloride.
Effect of 4-aminobenzenesulfonic acid concentration
The influence of the concentration of 4-aminoben-
zenesulfonic acid on the development of color was investi-
gated by treating varied concentrations (1.16 × 10-4 - 2.32 ×
10-3 M) of 4-aminobenzenesulfonic acid with 1.74 × 10-2 M
NaNO2 and 1.60 × 10-2 M HCl in a 25 mL standard volu-
Effect of NaNO2 concentration
The effect of the concentration of NaNO2 on the color
development was investigated in the range of 1.16 × 10-3 -
2.32 × 10-2 M. The absorbance (Fig. 2b) increased with in-

190 J. Chin. Chem. Soc., Vol. 54, No. 1, 2007
Rahman et al.
creasing concentration of reagent and became constant at
1.16 × 10-2 M, above which, up to 2.32 × 10-2 M, absorb-
ance remained constant. Therefore 1.74 × 10-2 M NaNO2
was chosen as an optimum value for the determination pro-
cess.
talol.
Analytical data and calibration graph
The calibration graph between the absorbance at 395
nm and the labetalol hydrochloride concentration was
found to be linear over the concentration range of 0.8-17.6
mg mL-1 with molar absorptivity and Sandell’s sensitivity
of 2.874 × 104 l mol-1 cm-1 and 0.013 mg cm-2 per 0.001
absorbance unit, respectively. The linear regression equa-
tion of the proposed method has been evaluated by the least
square treatment of the calibration data (n = 10). The linear
regression equation and correlation coefficient are A = 4.84
× 10-4 + 7.864 × 10-2 C where A is the absorbance and C is
the concentration of labetalol hydrochloride in mg mL-1 and
r = 0.9999, respectively, which indicated an excellent lin-
earity. Table 1 summarizes Beer’s law limit, linear regres-
sion equation, correlation coefficient, confidence limits
and standard deviations for slope and intercept at 95% con-
fidence level and variance, detection limit and quantitation
limit33 for the proposed method.
Effect of HCl concentration
The influence of the concentration of HCl on color
development has been investigated in the range of 1.60 ×
10-3 M - 1.84 × 10-2 M, and the results are shown in Fig. 2c.
The highest absorbance was obtained with 1.04 × 10-2
M
HCl; above this concentration, up to 1.84 × 10-2 M, the
absorbance remained constant. Therefore, the concentra-
tion of 1.44 × 10-2 M HCl was used in all measurements.
Effect of Na2CO3 concentration
The effect of the concentration of Na2CO3 was stud-
ied in the range of 5.60 × 10-3 - 5.04 × 10-2 M. As can be
seen from Fig. 2d, the absorbance of the colored product in-
creased with increasing concentration of Na2CO3 and re-
mained constant at 3.36 × 10-2 M. Further increase in con-
centration of the reagent up to 5.04 × 10-2 M caused no
change in the absorbance. Thus, 4.48 × 10-2 M Na2CO3 was
used as an optimum value for the determination of labe-
The statistical analysis of the calibration data has
made it possible to calculate error (Sc) in the determination
of a given concentration of labetalol hydrochloride using
the following equation:34
é
ê
ù1/ 2
ú
S0
b
1
(A- A)2
Sc =
1+
+
b2 (C -C)2
ê
ú
n
å
i
ê
ë
ú
û
i
where A and C are average values of the absorbance and
concentration, respectively, for n standard samples. The
plot of Sc versus concentration of labetalol hydrochloride
(Fig. 3) indicates that the minimum error is obtained when
the analysis is carried out at 8.40 mg mL-1. The value of Sc
can be used to establish the confidence limit at the selected
level of significance for the determination of unknown con-
centrations of labetalol hydrochloride using the relation Ci
± tSc. The results are presented graphically (Fig. 4) by plot-
ting the relative uncertainty (%DC/C) against the concen-
tration of labetalol hydrochloride35 at 95 and 99% confi-
dence levels for n-2 degrees of freedom. Thus, the confi-
dence limit can be established and the relative uncertainty
can be estimated directly on the concentration over the full
range of the concentration tested.
Fig. 2. Effect of the molar concentration of reactants:
(a) 4-aminobenzenesulfonic acid; (b) NaNO2;
(c) HCl; and (d) Na2CO3 with 16.0 mg mL-1
labetalol hydrochloride on the absorbance of
the colored product.
Specificity
The specificity of the proposed method was evaluated

Determination of Labetalol Hydrochloride
J. Chin. Chem. Soc., Vol. 54, No. 1, 2007 191
Table 1. Optical performance and regression characteristics of the proposed method
Parameters
max (nm)
Proposed method
l
Stability (h)
395
02
Beer’s law limit (mg mL-1)
0.8-17.6
2.87 ´ 104
Molar absorptivity (L mol-1 cm-1)
Linear regression equation
A = 4.84 ´ 10-4 + 7.86 ´ 10-2
C
Sa
1.02 ´ 10-3
2.35 ´ 10-3
9.99 ´ 10-5
2.30 ´ 10-4
0.9999
± tSa
Sb
± tSb
Correlation coefficient (r)
Detection limit (mg mL-1)
Quantitation limit (mg mL-1)
0.08
0.23
Sa and Sb are standard deviation of intercept and slope, respectively.
by estimating the amount of labetalol in the presence of
varying amounts of degraded products of labetalol such as
2-hydroxy-5-formylbenzamide and 1-methyl-3-phenyl-
propylamine, which were the major degradants of labe-
talol. It was found that 1-methyl-3-phenylpropylamine
did not interfere with the proposed method, whereas 2-hy-
droxy-5-formylbezamide interferes seriously.
for 2,2 and 5-day-time points, respectively, at room tem-
perature (25 1 °C). It was observed that stress by such
conditions did not cause significant degradation. There was
no change in the absorption spectra of the pure drug and
stressed (light, acid and base) sample solutions. The two re-
ported metabolites of labetalol such as O-sulfate (I, major)
and O-glucuronide (II, minor) (Scheme III) did not inter-
fere with the proposed method because of the denaturation
The various excipients commonly used in dosage
forms such as sodium stearyl fumarate, magnesium stearate,
starch, lactose and talc did not interfere with the proposed
method. The samples were stressed by light, acid and base
Fig. 4. Plot showing variation in the confidence limit
at (a) 95% confidence and (b) 99% confidence
level: relative uncertainty percentage (% DC/C)
versus concentration of labetalol hydrochlo-
ride.
Fig. 3. Plot of error (Sc) versus concentration of labe-
talol hydrochloride in the determination pro-
cess.

192 J. Chin. Chem. Soc., Vol. 54, No. 1, 2007
Rahman et al.
Scheme III Metabolites of labetalol: I by sulfation and II by glucuronidation process
CONH2
OH
CH3
N
H
OH
hepatic metabolism
CONH2
OH
-
OH
OSO2O
CH3
OH
CONH2
N
H
O
OH
CH3
COO
I
N
H
OH
II
of the phenolic group in these metabolites.36 The proposed
method is specific and selective for not giving reaction
with compounds, which lack a phenol group in the moiety.
recommended procedure. The % recovery RSD resulting
from the ruggedness chemist (tablet: 99.97 0.63%; cap-
sule: 100.06 0.58) and the developing chemist (tablet:
99.98 0.52%; capsule: 99.97 0.53%) were compared.
The % recovery and RSD results agreed well within the ac-
ceptable limits with permissible bias of all samples within
2.0%.
Robustness
The robustness of the proposed method relative to
each operational parameter was challenged. The opera-
tional parameters investigated were as follows:
· volume of 0.029 M 4-aminobenzenesulfonic acid,
1.5 mL ( 0.5 mL)
Accuracy and precision of the proposed methods
The accuracy and precision of the proposed method
was established by determining the content of labetalol in
pure and pharmaceutical formulations at three different
concentration levels (1.6, 8.0, and 16.0 mg mL-1). The
intra-day and inter-day precisions of the proposed method
in pure form was evaluated by carrying out five independ-
ent analyses at each concentration level within one day and
on five consecutive days (Table 2). In the same manner, the
intra-day and inter-day precisions were also performed in
drug formulations by measuring the amount of labetalol at
each concentration level (Table 3). The results presented in
Table 2 and 3 can be considered to be very satisfactory.
The validity of the proposed method was evaluated
by recovery studies using the standard addition method.
For this purpose, a known amount of reference drug was
added to formulated tablets and capsules at two different
concentration levels, and the nominal amount of drug was
determined by the proposed method. Each concentration
level was analyzed five times, and the results (Table 4)
· volume of 0.29 M NaNO2, 1.5 mL ( 0.5 mL)
· volume of 0.2 M HCl, 2.0 mL ( 0.5 mL)
· volume of 0.28 M Na2CO3, 4.0 mL ( 1.0 mL)
· cooling time of 10 min ( 3.0 min)
Under these conditions, a quality control sample solu-
tion containing 8.0 mg mL-1 (Lobet 100, Samarth Pharma)
of labetalol hydrochloride was assayed five times. The re-
sults obtained by the quality control sample represent good
recovery with acceptable bias of qL > 0.98 and qu < 1.02.
Ruggedness
The method ruggedness was evaluated by a second
analyst using a different spectrophotometer (Spectronic
20D+, Milton Roy Company, USA) and freshly prepared
standard sample solutions. The quality control tablet and
capsule samples of labetalol hydrochloride were analyzed
five times at one concentration level (8.0 mg mL-1) by the
ruggedness chemist and developing chemist following the

Determination of Labetalol Hydrochloride
J. Chin. Chem. Soc., Vol. 54, No. 1, 2007 193
Table 2. Intra-day and inter-day assays: Test of accuracy and precision of the proposed method in
pure form
Intra-day Assay
Amount, (mg mL-1)
Inter-day Assay
Amount, (mg mL-1)
SAEb
C.L.c
SAEb
C.L.c
Taken
Found ± SDa
Taken
Found ± SDa
1.6
1.601 ± 0.043
8.002 ± 0.042
16.014 ± 0.0430 0.019
0.019
0.019
0.053
0.052
0.053
1.6
8.0
1.601 ± 0.047
8.005 ± 0.047
16.011 ± 0.0490
0.021
0.021
0.022
0.059
0.058
0.061
8.0
16.0
16.00
a Mean for five independent analyses.
b SAE, standard analytical error.
c C.L., confidence limit at 95% confidence level and four degrees of freedom (t = 2.776).
Table 3. Intra-day and inter-day assays: Test of accuracy and precision of the proposed method in pharmaceutical formulations
Intra-day Assay Inter-day Assay
Amount (mg mL-1) Amount (mg mL-1)
Formulationsa
SAEb
C.L.c
SAEb
C.L.c
Taken
Found ± SD
Taken
Found ± SD
Tablet
Lobet 100
1.6
8.0
1.599 ± 0.043
7.997 ± 0.043
16.016 ± 0.045
0.019
0.019
0.020
0.054
0.053
0.056
1.6
8.0
1.604 ± 0.046
7.997 ± 0.051
16.003 ± 0.054
0.021
0.023
0.024
0.058
0.063
0.067
(Samarth Pharma)
16.00
16.00
Capsule
Normadate 50
(Glaxo Smithkline)
1.6
8.0
1.601 ± 0.045
7.997 ± 0.043
16.011 ± 0.043
0.020
0.019
0.020
0.055
0.053
0.053
1.6
8.0
1.601 ± 0.047
8.005 ± 0.047
15.998 ± 0.051
0.021
0.021
0.023
0.059
0.058
0.063
16.00
16.00
a Mean for five independent analyses.
b SAE, standard analytical error.
c C.L., confidence limit at 95% confidence level and four degrees of freedom (t = 2.776).
Table 4. Standard addition method: Evaluation of the validity of the proposed method for the recovery of
labetalol hydrochloride
Concentration (mg mL-1)
Recovery ± RSDa
(%)
Formulationsa
SAE
C.L.
Taken
Added
Found ± SDa
Tablet
Lobet 100
4.8
8.0
3.2
8.0
7.992 ± 0.041
16.014 ± 0.046
99.90 ± 0.52
100.09 ± 0.27
0.018
0.019
0.051
0.053
(Samarth Pharma)
Capsule
Normadate 50
(Glaxo Smithline)
4.8
8.0
3.2
8.0
7.997 ± 0.043
16.011 ± 0.043
99.97 ± 0.53
100.07 ± 0.27
0.019
0.019
0.053
0.053
a Mean for five independent analyses.
show good recovery with low SD and RSD. No interfer-
ence from the common excipients was observed.
using point and interval hypothesis tests.37 The student’s t
and F-values at 95% confidence level did not exceed the
tabulated t- and F-values, confirming no significant differ-
ences between the performance of the proposed method
and the reference method (Table 5). Once it is ensured that
the proposed method is not significantly different from the
The applicability of the proposed method has been
tested for the determination of labetalol in drug formula-
tions. The results of the proposed method were compared
with those obtained by Bilal’s spectrophotometric method25

194 J. Chin. Chem. Soc., Vol. 54, No. 1, 2007
Rahman et al.
Table 5. Point and interval hypothesis tests: Evaluation of the applicability of the proposed method
with the reference method at 95% confidence level
Proposed method Reference method
t & F-
valuesb
Formulationsa
c
qL
c
qU
Recovery
%
RSDa
Recovery
%
RSDa
%
%
Tablet
Lobet 100
99.97
0.63
99.89
99.98
0.81
0.86
t = 0.211
F = 1.626
0.991 1.011
0.987 1.014
(Samarth Pharma)
Capsule
Normadate 50
(Glaxo Smithkline)
100.030
0.52
t = 0.148
F = 2.722
a Mean for five independent analyses.
b Theoretical t-value (n = 8) and F-value (n = 4, 4) at 95% confidence level are 2.306 and 6.39,
respectively.
c In pharmaceutical analysis, a bias, based on recovery experiments, of ± 2% is acceptable.
Table 6. Comparison of the proposed method with existing spectrophotometric methods for the determination of
labetalol hydrochloride in drug formulations
lmax
(nm)
Beer’s law limit
(mg mL-1)
RSD
(%)
Molar absoptivity
(l mol-1 cm-1)
Reagents
References
2,6-dichloroquinone chlorimide
Folin-Ciocalteu’s reagent
670
650
510
530
685
5-25
8-40
2.5-15
50-250
-
-
1.39 ´ 104
5.88 ´ 103
2.09 ´ 104
1.05 ´ 103
[24]
[24]
[24]
[24]
[26]
-
4-aminophenazone
-
Ferric ammonium sulfate
-
p-N,N-dimethylphenylenediamine
hydrochloride & sodium hypochlorite
3-methyl 2-benzothiazolinone hydrazone
hydrochloride & ceric ammonium sulfate
Suprachen violet 3Ba
0.35-0.52
545
-
0.35-0.52
[26]
565
600
395
-
0.35-0.52
0.64
[26]
[27]
Wool fast blue BLa
1-6
Diazotized 4-aminobenzenesulfonic acid
0.8-17.6
0.34-0.52
2.87 ´ 104
This work
a Extractive method.
reference method, it is necessary to check the bias of each
drug sample based on recovery experiments using the inter-
val hypothesis test. In pharmaceutical analysis, The Cana-
dian Health Protection Branch has recommended a bias of
± 2% with a lower limit (qL) of 0.98 and an upper limit (qU)
of 1.02 in pharmaceutical analysis.38 It is evident from Ta-
ble 5 that the true bias of all samples is < ± 2%. The interval
hypothesis tests have confirmed that the accuracy and pre-
cision are within the acceptable limits and indicated no sig-
nificant differences between the performances of the pro-
posed method with the reference method at 95% confi-
dence level.
(Table 6). The molar absorptivity of the proposed method
is higher than that of other methods, indicating its great
sensitivity. It has the advantage of determining labetalol as
low as 0.8 mg mL-1. Moreover, the proposed method is fast
with respect to analysis time and directly applicable to the
drug sample without prior extraction needed in other meth-
ods.26,27 The method is versatile and useful due to inde-
pendence from pH and a high tolerance limit from common
excipients present in drug formulations.
CONCLUSION
The proposed method for the determination of labe-
talol hydrochloride in drug formulations was compared fa-
vorably with other existing spectrophotometric methods
The proposed method is a stability-indicating assay
method for the determination of labetalol in pure form and

Determination of Labetalol Hydrochloride
J. Chin. Chem. Soc., Vol. 54, No. 1, 2007 195
matogr. 2001, 15, 393.
in drug formulations. Point and interval hypothesis tests
proved that the proposed method has acceptable bias for all
pharmaceutical samples that lie within 2.0%. The results
demonstrate that the proposed method is accurate, precise,
linear, specific and robust for the determination of labetalol
hydrochloride in tablets and capsules and thus can be ex-
tended for the routine quality control analysis of the drug in
the pharmaceutical industry, hospitals and research labora-
tories.
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The authors are grateful to Aligarh Muslim Univer-
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trial Research (CSIR), New Delhi, India, to Dr. Syed
Najmul Hejaz Azmi as Research Associate (Award No.
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