ISSN-1996-918X Pak. J. Anal. Environ. Chem. Vol. 12, No. 1 & 2 (2011) 55-60 Spectrophotometric Determination of Primaquine by Coupling with Diazotized p-Nitroaniline Application to Pharmaceutical Formulations
Ferial Mahmood El-Samman, Asma Natiq Al-Irhayim*
Department of Chemistry, College of Science, Mosul University, Mosul, Iraq
Received 06 July 2010, Revised 12 August 2010, Accepted 10 October 2011
-------------------------------------------------------------------------------------------------------------------------------------------- Abstract A simple spectrophotometric method for the determination of primaquine phosphate has been developed. The method is based on the coupling reaction of primaquine phosphate in acidic medium with diazotized p-nitroaniline, to form a yellow-orange water-soluble azo dye that shows maximum absorption at 482 nm. Beer’s law is obeyed over the concentration range 5-300 µg of primaquine phosphate in a final volum of 25 ml, i.e., 0.2-12 ppm with a molar absorptivity of 6.6104 l mol-1 cm-1 and Sandell’s sensitivity index of 0.0068 µg.cm-2, a relative error of -0.63 to +0.32% and a relative standard deviation of ±0.18 to ±2.8%, depending on the concentration level. Interferences due to excipients have been examined. The proposed method has been applied to the assay of primaquine phosphate in pharmaceutical formulations (tablets). Keywords: Primaquine; diazotized p-nitroaniline; spectrophotometry.
-------------------------------------------------------------------------------------------------------------------------------------------- Introduction Primaquine
involves the dissolution of the sample in anhydrous
Pentane-1,4-diamine] is an antimalarial drug that is
acetic acid with gentle heating. When cooled, the
very important for the radical cure of relapsing
sample is titrated against perchloric acid [11], and
vivax or ovale malaria and it eliminates tissue
the end point is determined potentiometrically.
infection. It is a member of the 8-aminoquinoline
group of drugs and is often administered in
been described for the determination of primaquine
phosphate. Some of these methods are comprised
There are various analytical procedures for
of coupling of primaquine phosphate with
the assay of Primaquine, an HPLC [3, 4],
diazotized reagents [12, 13] to form an intensely-
facilitating metabolic and pharmacokinetic studies
coloured azo dye. Other colorimetric methods are
[5]. A capillary electrophoresis method has been
based on the reaction of primaquine with chloanily,
developed that allows the separation and
tetracyanoethylene [14, 15, 16] are reported. Some
pharmaceutical formulations [6]. Other methods
of these methods are time-consuming [12, 14] less
such as voltammetry [7], gas chromatography-
sensitive [16], require organic solvents [12], buffer
mass spectrometry [8], and ultra-performance
liquid chromatography [9] (UPLC) have also been
proposed. The British Pharmacopoeia method [10]
Pak. J. Anal. Environ. Chem. Vol. 12, No. 1 & 2 (2011)
the final solution was within the working range
primaquine has been worked out, depending on
p-nitroaniline to form a highly coloured dye that
Procedure
has proved successful for the determination of
primaquine in pharmaceutical preparations.
An aliquot of standard solution containing
5-300 µg of primaquine phosphate was transferred
Experimental
to a 25 ml volumetric flask. 2 ml of diazotized p-
Apparatus
nitroaniline (5 mM) solution is added followed by
2 ml of 0.2 M acetic acid solution and diluted to
Absorption spectra are carried out using
the mark. The solution was mixed thoroughly and
Shimadzu UV-Visible recording Spectrophoto-
the absorbance was measured at 482 nm against
the reagent blank within 2 h using 1-cm cells.
Results and Discussion
carried out on a single beam Spectrophotometer
Absorption spectra
dye formed as a result of mixing a dilute aqueous
Philips PW 9420 pH-meter with a combined glass
solution of primaquine phosphate and diazotized p-
nitroaniline in acidic medium. The azo dye formed
shows maximum absorption at 482 nm (Fig. 1), in
Reagents
contrast to the reagent blank. The colour is formed
immediately and is stable for at least 120 minutes.
Chemicals used are of analytical reagent grade. The calibration graph is linear over the range 0.2 -
Working primaquine phosphate solution, 12 ppm. The apparent molar absorptivity has been
Diazotized p-nitroaniline reagent solution,
.) iv 0.200
5mM: A 0.1727 g of p-nitroaniline is dissolved in
about 50ml distilled water. Then 20 ml of 1 M HCl
is added and solution is heated, the clear mixture is
then transferred to a 250 ml volumetric flask and is
cooled to 0-5oC in an ice-bath. 8.65 ml of 1%
NaNO2 is added and the mixture is stirred
400.0 50.0 (nm/Div.) 650.0
vigorously. After 5 min the solution is made up to
Wavelength, nm
volume in 250 ml volumetric flask with cold water.
Figure 1. Absorption spectra.
The solution is stored in a brown bottle in a
refrigerator and is stable for at least one week.
Effect of surfactants
Primaquine tablets solution: Four tablets
The effect of different types and amounts
were powdered accurately weighted and the weight
of surfactants (cetyltrimethylammonium bromide
of the powder equivalent to one tablet was
(cationic surfactant), sodium dodecyl sulfate
dissolved and transferred into a 100 ml calibrated
(anionic surfactant) and Tween 80 on the colour
flask and made up to volume with water. The
intensity of the yellow dye has been examined and
solution was filtered and the clear filtrate used for
the results showed that the addition of surfactants
the determination. An appropriate volume of the
gives no significant effect. Therefore, it has been
sample solution was diluted further with water so
recommended to eliminate the use of surfactants in
that the concentration of primaquine phosphate in
Pak. J. Anal. Environ. Chem. Vol. 12, No. 1 & 2 (2011) Effect of acid added Table 2. Effect of different amounts of bases on absorbance of Azo-dye. Absorbance /ml of Base used 0.1 M of Base
examined for their effect on the colour intensity of
the azo dye. The experimental order of the acid
(sample+acid+diazotized reagent or sample +
diazotized reagent + acid) added have no effect on
the intensity of the coloured azo dye Table 1.
Table 1. Effect of different amounts of acids on absorbance of Azo-dye. Absorbance/ ml of Acid used 0.1 M of Acid Choice of diazotized reagent and amount
Diazotized p-nitroaniline reagent has been
selected in this study because of following reasons;
its the strongest diazonium electrophile ever used
(due to the presence of the strong electron-
withdrawing nitro group), the strongest colour
contrast observed in its azo dye production (due to
the presence of the p-nitro resonating group), the
most sensitive diazo-coupling reaction diazotized
p-nitroaniline reagent can give with aromatic
components [17, 18] and the colour of the
diazotized reagent solution is faint yellow thus
giving lower blank values. The effect of the
Effectofbase
amount of diazotized p-nitroaniline reagent on the
maximum absorbance of the dye formed has been
investigated and the results are illustrated in
various bases (NaOH, KOH, Na2CO3, NaHCO3,
investigated for their effect on the colour intensity
Table 3. Effect of amount of diazo reagent solution on absorbance.
of the azo dye. The colour of the azo dye changes
from yellow-orange in acidic media to orange then
Primaquine Absorbance/ ml (5 mM) diazotized reagent phosphate
to yellowish-brown and also becomes turbid as the
amount of base increases (then pH of final reaction
mixture is nearly five or more). In addition the
blank changes from colourless in acidic media to
medium because of strong colour contrast and
better expected selectivity of determination
because most compounds are activated towards
2 ml of 5 mM diazotized reagent has been
selected for the procedure, since it gives the
4 ml of 0.1 M of the acid) is selected for the
Pak. J. Anal. Environ. Chem. Vol. 12, No. 1 & 2 (2011) Effect of time on color development
The effect of time with different amounts
of primaquine phosphate has been investigated for
Nature of the dye
maximum production of the dye and longer
stability period Table 4. The maximum absorbace
reading is given immediately and remains constant
indicates that the dye has a composition of 1:1
for at least 120 minutes, independent of
primaquine to diazotized p-nitroaniline reagent, the
structure of the azo dye is represented in scheme 1.
Table 4. Effect of time on colour development. Absorbance/ g of primaquine phosphate Scheme-1. Yellow-orange azo dye Accuracy and precision
primaquine has been determined at three different
concentrations Table 6. The low value of relative
error and relative standard deviation shows that the
Effect of organic solvents Table 6. Accuracy and precision of the method. Relative* Amount of primaquine Relative*
optical properties of the azo dye has been tested.
standard Phosphate taken (g)
The reaction mixture has been diluted using
deviation%
different organic solvents and the data are shown
Table 5. Effect of various organic solvents on the absobance of the azo dye.
Absorbance *Average of five determinations Effect of interferences
In order to assess the possible analytical
applications of the present proposed method, the
interfering effect of foreign substances on the
determination of 100 µg of primaquine phosphate
is shown in Table 7. It is evident from the table
that the proposed method is quite selective.
Pak. J. Anal. Environ. Chem. Vol. 12, No. 1 & 2 (2011) Table 7. Effect of excipients on the determination of different
using the present method in the determination of
amount of primaquine phosphate. Interferent Recovery % added g Table 9. Comparison of the methods and experimental t-test values. Recovery % British Drug (tablets) Present pharmacopeia method method
The results show no significant difference
Application of the method
between the present method and the standard
To test the applicability of the present
method it has been applied to the determination of
primaquine phosphate in to drugs Table 8.
some of analytical variables obtained from the
present method and another spectrophotometric
Table 8. Determination of primaquine phosphate in tablets. g Primaquine Manufacturing Recovery* Table 10. Comparison of the methods. phosphate Present measured Literature Analytical parameters Present method
contains primaquine phosphate 26.3 mg which is
From the results, it can be shown that good
agreement has occurred between the amount of
primaquine phosphate present and that measured
Comparison of the methods and t-test
and British pharmacopeia standard method [10] for
the determination of primaquine phosphate in two
* Sulaiman S.T. and Amin D., Inteern. J. Environ. Anal. Chem.18
drugs, is based on the t-test to show the ability of
Pak. J. Anal. Environ. Chem. Vol. 12, No. 1 & 2 (2011)
M. L. P. M. Arguelho, M. V. B. Zanoni and
over the published methods that it occurs at room
N. R. Stradiotto, Anal. Lett., 38 (2005) 1415.
temperature and not specific for a particular.
I. Brondz, A. B. Fialkov, and A. Amirav, J. Conclusion
V. G. Dongre, P. P. Karmuse, P. R. Pilla and
A. Kumar, J. of Pharm. and Biomed.
precise, and accurate determination of primaquine
British Pharmacopeia on CD-ROM (System
phosphate with an application to pharmaceutical
analysis. The short analysis time and low costs are
the main advantages of this method for routine
A. D. Skoog, and D. M. West, Fundamental of Analytical Chemistry, (Rinehart and
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