Talanta 47 (1998) 639 643

Visible spectrophotometric and first-derivative UV

spectrophotometric determination of rifampicin and isoniazid in
pharmaceutical preparations
S.A. Benetton, E.R.M. Kedor-Hackmann *, M.I.R.M. Santoro, V.M. Borges
Departamento de Farmacia, Faculdade de Ciencias Farmaceuticas, Uni6ersidade de Sao Paulo, Caixa Postal 66355, CEP 05389 -970,
Sao Paulo, Brazil
Received 6 October 1997; received in revised form 2 February 1998; accepted 26 February 1998

Abstract

Two methods are described for the determination of rifampicin and isoniazid in mixtures by visible spectrophoto-
metry and first-derivative ultraviolet spectrophotometry. The absorbance at 475 nm in buffer solution pH 7.4 was
employed to determine rifampicin after applying the three-point correction technique between 420 and 520 nm, while
the amplitude of the first-derivative spectrophotometric spectrum at 257 nm in HCl 0.012 M was selected for the
determination of isoniazid. The methods are rapid, simple and do not require any separation step. The recovery
average was 99.03% for rifampicin and 100.01% for isoniazid. The methods were applied to determine the two
compounds in commercial capsules and compared with the official method of the USP XXIII with good agreement
between the results. 1998 Elsevier Science B.V. All rights reserved.

1. Introduction introduced to improve patient acceptability and

Since isoniazid (INH) and rifampicin (RIF)
form one of the most effective antituberculosis
regimens used in many countries, considerable
effort has been spent on improving the efficacy of
this therapy [1]. For most patients, regimens that
contain these drugs can successfully be completed
in 6 months if pyrazinamid is included in the
regimen for the first 2 months. The most serious
problem with tuberculosis therapy is patient non-
adherence to the prescribed regimens and com-
bined formulations of RIF and INH were
* Corresponding author. Fax: +55 118154418.
compliance. The combined formulation is official
in the United States Pharmacopeia with the name
rifampin and isoniazid capsules [2].
Although some procedures have been described
for the assay of either RIF or INH in pharmaceu-
tical preparations and in biological fluids, there
are only a few methods reported to be able to
analyse both drugs in combination [3,4]. These
methods use high performance liquid chromatog-
raphy (HPLC) and, although they are sensitive,
they are laborious and expensive. The compendial
method available (USP XXIII) for the assay of
RIFINH mixture in capsules employs a HPLC
method for the determination of RIF and a titri-

0039-9140/98/$ - see front matter 1998 Elsevier Science B.V. All rights reserved.
PII S0039-9140(98)00111-8
640 S.A. Benetton et al. / Talanta 47 (1998) 639643
metric method, which requires a separation step,
for the determination of INH [2].
The aim of this work was to investigate the
utility of derivative spectrophotometry in the as-
say of RIF and INH in combination in pharma-
ceutical preparations without the necessity of
sample pre-treatment. Derivative spectrophoto-
metry [5] is a useful technique for the suppression
of additive interference, and it has been used
extensively for the simultaneous determination of
substances in mixtures [6,7]. Two methods were
developed and validated in this work: a visible
spectrophotometric method using the three-point
correction technique [8] for the determination of
RIF and a derivative ultraviolet spectrophotomet-
ric method for the determination of INH. The
methods had sufficiently good accuracy and preci-
sion and permitted a simple and time-saving assay
of INH and RIF in mixtures.
2. Experimental
2.1. Apparatus
A Hewlett-Packard 8453 diode-array spec-
trophotometer fitted with a HP 845 UV-Visible
ChemStation and a Hewlett-Packard 600 printer
were used for all the measurements and treatment
of data.
Chromatography was performed using a CG
solvent delivery pump (model 480-C) and a CG
variable UV detector set at 254 nm connected to a
CG integrator (model CG-200) (Instrumentos Ci-
entficos CG, Sao Paulo, Brazil). The system was
equipped with a Rheodyne 7125 injection valve
fitted with a 20 ml loop. The analytical column
was a Waters Nova-Pak C8 (3.9150 mm, 5
mm) column. The chromatographic conditions
were the same described on the USP XXII under
rifampin assay conditions [2]. A Digimed DM21-
V6 pH meter was used for the determination of
the end point in the titration of INH.
2.2. Materials and solutions
Rifampicin and isoniazid were kindly donated
by a pharmaceutical industry and used without
further purification. All solvents and reagents
were of analytical grade. Phosphate buffer solu-
tion pH 7.4 was prepared according to the British
Pharmacopoeia 1993 [9]. The commercial phar-
maceutical formulations used in this work (cap-
sules) contained 300 mg of rifampicin and 200 mg
of isoniazid.
2.3. Procedures
2.3.1. Visible spectrophotometry
Standard solutions containing known quantities
of RIF (2070 mg ml 1) in phosphate buffer pH
7.4 were prepared from a methanolic stock solu-
tion for the calibration curve.
The commercial capsules were emptied in a
glass mortar and an amount of powder equivalent
to 125 mg of RIF was accurately weighed out into
a 100 ml amber volumetric flask. About 50 ml of
methanol were added, sonicated for 5 min and the
volume was made up with the same solvent. The
methanolic solution was filtered through a What-
man No. 1 filter paper and further dilutions of the
appropriate aliquots were made in phosphate
buffer pH 7.4 to obtain a final solution containing
50 mg ml 1. The absorbance of both the sample
and the standard solutions were measured at 475
nm after applying the three-point correction tech-
nique between 420 and 520 nm. Phosphate buffer
pH 7.4 was used in the reference cell.
2.3.2. First-deri6ati6e ultra6iolet
spectrophotometry
Standard solutions containing known quantities
of INH (525 mg ml 1) in HCl 0.012 M were
prepared for the calibration curve.
The commercial capsules were emptied in a
glass mortar and an amount of powder equivalent
to 125 mg of INH was accurately weighed out
into a 100 ml amber volumetric flask. About 50
ml of methanol were added, sonicated for 5 min
and the volume was made up with the same
solvent. After filtration through a Whatman No.
1 filter paper, a 5 ml aliquot was transferred to a
50 ml amber volumetric flask and diluted to vol-
ume with water. A 2 ml aliquot was transferred to
a 25 ml amber volumetric flask and diluted to
volume with HCl 0.012 M. The final concentra-
 S.A. Benetton et al. / Talanta 47 (1998) 639643 641

Fig. 1. Absorption spectra of (A) isonazid (20 mg ml 1) and (B) rifampicin (30 mg ml 1) in (a) HCl 0.012 M and (b) phosphate
buffer solution pH 7.4.

tion was 10 mg of INH ml 1. The amplitude of
the first-derivative of both the sample and the
standard solutions were measured at 257 nm. A
solution of HCl 0.012 M was used in the reference
cell.
The methods were validated as to precision
(reported as the relative standard deviation, RSD
%), linearity (evaluated by regression analysis),
and accuracy. Accuracy was determined by recov-
ery studies performed according to the USP
XXIII guidelines [2]: the methods were applied to
artificial samples which contained amounts of an-
alyte both above (120%) and below (80%) the
normal levels expected in the samples. The accu-
racy was then calculated from the test results as
the percentage of analyte recovered by the assay.
3. Results and discussion
3.1. Method de6elopment
The influence of pH on the absorption spectra
of RIF and INH was studied between pH 7.4 and
1.7. The pH value slightly affected the absorption
spectra of RIF but exerted a profound influence
on the quality and shape of the INH spectra (Fig.
1a, b). INH absorption spectra presented a shift
642 S.A. Benetton et al. / Talanta 47 (1998) 639643
Fig. 2. First derivative spectra of (A) isonazid (20 mg ml 1) and (B) rifampicin (30 mg ml 1) in HCl 0.012 M.
to shorter wavelength as well as narrower and
better-defined absorption spectra as the pH
value of the medium decreased. The absorption
spectrum of INH was completely overlapped
with the spectrum of RIF in both acidic and
alkaline conditions. Therefore, the determination
of INH in the presence of RIF cannot be per-
formed by direct absorbance measurements and
derivative spectrophotometry was then em-
ployed. The improved resolution of overlapping
absorption bands and the discrimination in fa-
vour of narrow bands against broader bands,
which are the principal characteristic of deriva-
tive spectrophotometry, can result in the com-
plete elimination of specific interference from the
co-formulated compound (Fig. 2). The pH-in-
duced shift of INH spectrum was exploited in
this work and the best results for the analysis of
INH by first order ultraviolet derivative spec-
trophotometry were obtained in HCl 0.012 M
(pH 2.0), because the spectrum of INH is nar-
rower in this medium than under less acidic
conditions. The height (amplitude) of a deriva-
tive spectrum depends not only on the height of
the normal spectrum but also on its width: for a
given peak height, narrower peaks give larger
derivative amplitudes. Therefore, the determina-
tion of INH was carried out by measuring the
amplitude of the first-derivative spectrum of
INH in HCl 0.012 M at 257 nm (zero-crossing
wavelength of rifampicin).
In Fig. 1a it also can be seen that the absorp-
tion of RIF showed a large peak at 475 nm that
can be used for direct absorbance measurement.
However, the stability of RIF is very weak un-
der these acid conditions (stable for less than 15
min) and the determination of RIF was carried
out in phosphate buffer solution pH 7.4 (Fig.
1b). As the determination of RIF in capsules
containing RIFINH mixtures by direct visible
spectrophotometry showed a small but system-
atic positive error, the three-point correction
technique was employed in an attempt to avoid
these biased results. The recovery tests showed
improved results after the application of that
correction technique.
3.2. Visible spectrophotometry (RIF analysis)
The precision (RSD %) of the results was of
0.56% as determined on ten replicate measure-
ments of a commercial sample. The linearity of
response, at a concentration range from 20 to
70 mg ml 1, was evaluated by regression analy-
sis and the regression equation (Y=0.0124X +
0.0026) presented a correlation coefficient of
0.9999. The average percent recovery determined
in the recovery test for RIF was 99.03%.
 S.A. Benetton et al. / Talanta 47 (1998) 639643
Table 1
Data obtained from the analysis of a commercial sample
containing isoniazid (200 mg) and rifampicin (300 mg) in
combination
Commercial sample Percentage of the declared amount
(capsules)
New methoda USP XXIII
(%) methodb (%)
Isoniazid 95.869 0.53 95.02
Rifampicin 98.329 0.34 97.13
a
Mean value of ten determinations9 95% confidence interval.
b
Mean value of three determinations.
3.3. First-deri6ati6e ultra6iolet spectrophotometry
(INH analysis)
The precision (RSD %) of the results was of
0.78% as determined on ten replicate measure-
ments of a commercial sample. The absorption
spectrum of INH was completely overlapped with
the spectrum of RIF (Fig. 1a). As the accuracy of
absorbance measurements is low at high ab-
sorbance values, the addition of the absorbance of
the two compounds limited the range of work for
the analysis of INH by first-order derivative UV
spectrophotometry (5 25 mg ml 1). The linearity
of response was evaluated by regression analysis
and the regression equation (Y = 0.121X 0.003)
presented a correlation coefficient of 0.9999. The
average percent recovery determined in the recov-
ery test for INH was 100.01%.
3.4. Comparison of the de6eloped methods with
the USP XXIII methods
The commercial rifampicin capsules co-formu-
643
lated with isoniazid were also assayed by the USP
XXIII official methods and the results compared
with those obtained using the developed methods.
The results can be seen in Table 1.
4. Conclusion
It was concluded that first-derivative UV spec-
trophotometry is suitable as a rapid alternative to
the official USP XXIII titration method for isoni-
azid in combination with rifampicin in pharma-
ceutical preparations. Rifampicin can also be
determinated with good accuracy and precision by
the visible spectrophotometric method employing
the three-point correction technique. The short
analysis time and low costs are the main advan-
tages of these methods for routine analysis.
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