INTRODUCTION:
Organic compounds containing hydrazine functions find
many commercial and therapeutic applications. Isoniazid (1), Hydralazine hydrochloride
(2) and Dihydralazine sulphate (3), are the commercial drugs containing hydrazine
function. Whereas the former is used for the Anti-bacterial, Antifungal and Anti-tubercular[1,2] while
other two are used for the control of hypertension, depression, bacterial infections,
and for the modification of cardiac rate and rhythm.
Figure 1:
Isoniazid Figure 2: Hydralaizine
hydrochloride
Figure 3: Dihydralazine sulphate
The analysis of these drugs for their
active ingredient content is highly desirable for quality assurance of drugs
and to maintain uniform therapeutic
standards with respect to both dosage form and quality. Thus their rapid
and accurate determination in commercial formulations is of immense significance.
Several methods have been reported for the determination of these drugs which
includes spectrophotometry[3-15], spectrofluorimetry[16],
gas chromatography-mass spectrometry (GC-MS)[17-21],
high-performance liquid chromatography (HPLC)[22-24], liquid
chromatography-mass spectrometry (LC-MS)[25,26], chemiluminescence[27],
capillary electrophoresis[28,29], voltammetry[30-32], chromatography[33,34],
titrimetric[35] and optical sensor technology[36].
Although chromatography is considered as a dominant
analytical technique for the analysis of drugs but the methods based on these techniques
require good experimental skill to obtain reproducible and reliable data.
Hence, the development of simple, time-saving, rugged and economic procedures
is of crucial importance. The polarographic methods are advantageous in the
above analysis because the determinations can be done even at larger dilutions and
with smaller volumes of solutions without any interference from inert carriers
commonly present in their formulations but have attracted little attention. The
author has been able to work out a sensitive pulse polarographic method in non
aqueous media to analyze listed drugs in their commercial formulations.
Advantage
has been taken of the transformation of the hydrazine function of each drug
compound with carbon disulphide in dimethylformamide (DMF) to form corresponding
drug dithiocarbazate derivatives and the reaction of later at dropping mercury electrode
(DME) in the presence
of pyridinium perchlorate supporting electrolyte to yield analytically useful diffusion controlled waves and peaks at -1.10, -1.28 and -0.94 (vs SCE) respectively
in developing the proposed method. The
results have been calculated on the basis of calibration graph drawn between
drug concentration and current. The
proposed method allowed analysis in non aqueous media is advantageous in that these
drugs are not freely soluble in water and undergo hydrolysis and decomposition
in aqueous acidic/alkaline solutions which are commonly encountered as buffers
or supporting electrolytes in polarography
METHODS AND MATERIALS:
The analytical standards of high purity of Hydralazine hydrochloride (Genex Pharma, % age purity ≥ 96%), Dihydralazine sulphate (Sigma-Aldrich, % age
purity ≥ 99%) and isonicotinic acid
hydrazide (Isoniazid, Sigma-Aldrich, % age
purity ≥ 98%) have been procured. The
purity of each compound has been checked by known methods [37-39]. Two
formulations of Hydralazine hydrochloride viz. apresoline and corbetazine and
two formulations of Dihydralazine sulphate viz. adalphane and nepresol and four
formulations of isoniazid viz. solonex, R-cinex, isonex-300, isonex Fort-300 have
been procured from local market.
Dimethylformamide (BDH) has been purified by storing
it over anhydrous sodium carbonate (A.R) for two days. The solvent decanted
off, distilled and the fraction distilling at 148.5-149.5o has been
collected in coloured bottles. Acetonitrile (E. Merck) has been distilled twice
from phosphorus pentaoxide (5gl-1). Carbon disulphide (Baker
analysed, 100% estimated chromatographically) has been used as received.
Pyridinium perchlorate, 0.01M in DMF has been prepared by dissolving 0.1784g of
pyridinium perchlorate in 100 ml of dimethylformamide. Triton X-100: 0.02% in
dimethylformamide has been used as suppressor.
All
polarographic measurements have been made with a polarographic analyser (model
CL-362, Elico, Hyderabad, India). The electrode system consisted of DME as
working electrode, saturated calomel electrode (SCE) as reference electrode,
and platinum as an auxiliary electrode.
General Procedure:
Preparation of calibration graphs for pure drug
compounds:
A
calibration curve has been prepared under optimized conditions. Stock standard solution (10-5M)
of each pure drug compound has been prepared in DMF. Aliquots (0.1-1.0ml) of each
standard drug solution has been taken in polarographic cell and mixed with 1
drop (~50 μl) of carbon
disulphide and pyridinium perchlorate (20ml, 0.01M in DMF), Triton X-100 (2ml,
0.002% in DMF) and the final volume has been made to 25ml with DMF. Nitrogen
gas has been bubbled through the solution for 5 minutes and polarograms of each solution has been recorded at room temperature
(23±1)°C with the following instrumental parameters : initial potential = 50mV,
drop time = 0.5s, pulse amplitude = 50mV, and scan rate = 3mV s-1. A
calibration graph has been constructed by plotting the peak current (μA)
versus the concentration of drug compound (μg mL-1) and is
shown in Fig 5, 6 and 7. The
results of determination have been given in Tables 3, 4 and 5.
With a view to ascertain the diffusion-controlled
nature of the electrode reaction, polarograms of standard solutions of each
drug and at a fixed concentration but at various heights of mercury column have
been recorded in usual way. A typical plot showing linear relationship between
id and ÖhHg is
represented in Fig. 4. The values of idÖhHg are given in Table 2(a), 2(b).
Drug analysis:
Two formulations of Hydralazine hydrochloride viz. apresoline
containing 50mg per tablet and corbetazine containing 25mg per tablet active
ingredient, four formulations of isoniazid viz. R-cinex, solonex, Isonex Forte-300
each containing 300mg per tablet and Isonex-100 containing 100mg per tablet
active ingredient, two formulations of Dihydralazine sulphate viz. adelphane
containing 10mg and Nepresol containing 25mg per tablet active ingredient have
been used. A single large sample of each formulation has been weighed and
shaken with dimethylformamide and filtered. The residue has been washed 2-3
times with 4-5ml instalments of respective solvent. The washings and filtrate
have been diluted to known volume with same solvent. Aliquots have been taken
into polarographic cell and processed for analysis in the same manner as
described above for pure compounds. The results of analysis have been
summarized in Tables 6, 7 and 8.
RESULTS AND DISCUSSION:
In the course of present investigations on the
determination of hydrazine drugs as dithiocarbazates (through reaction with
carbon disulphide), the author has found that they are very reactive and
undergo electrochemical reaction at DME yielding analytically useful diffusion
controlled peaks which possess the characteristics required for adoption for
quantitative use And hence applied to the pulse polarographic analysis of
listed hydrazine drugs. Although, drugs are not directly involved in the
method, each is stoichiometrically related to the amount of dithiocarbazates.
The electrode-reaction at DME is diffusion controlled is evident from the
linear relationship obtained between diffusion current (id) and
square root of mercury column height (ÖhHg)
(Fig. 4, Table 2a and 2b).
The
reaction taking place at the mercury electrode is supposed to involve the
formation of mercurous dithiocarbazate undergoing one electron change and the
same is established by
making use of the equation of the polarographic wave[27,
28].
E = E1/2 + 0.0591/n (log (id-i)/i)
It
follows from the equation that plot of (log (id-i)/i) against
the corresponding potential (E), a straight line should be obtained with a
slope of 0.0591/n for a reversible reaction. The value of n, the number of
electrons taking part in the reversible reaction, can be determined. In the
present case a straight line with slope 0.0797 is obtained indicating n = 0.74
(~1).
The analysis has been accomplished by DPP using linear
calibration plots. Calibration graphs prepared between concentration of each
drug compound taken and diffusion/peak current measured in terms of peak height
(DPP) were linear upto 40, 48, 40μg
ml-1 respectively for isoniazid, Hydralazine hydrochloride and dihydralazine
sulphate. The results recorded in Table 3, 4 and 5 shows that the above
compounds in the ranges 2.5-40, 3-48 and 2.0-40μg could be determined with
maximum relative standard deviation's (RSD's) of 0.6, 0.6 and 0.8% respectively.
Table 3: Pulse polarographic determination of Isoniazid .
|
Amount taken, μg
|
DPP
|
|
Mean peak current, ip, μA
|
Amount found*, μg
|
|
3
|
3.13
|
2.98+0.02
|
|
6
|
6.24
|
5.94+0.04
|
|
12
|
12.64
|
12.04+0.05
|
|
24
|
25.39
|
24.18+0.05
|
|
48
|
50.34
|
47.94+0.06
|
* Values are mean of three determinations with standard
deviation (+)
Table 4: Pulse polarographic determination of Hydralazine
hydrochloride
|
Amount taken, μg
|
DPP
|
|
2.5
|
3.89
|
2.48+0.02
|
|
5.0
|
7.78
|
4.96+0.04
|
|
10.0
|
15.71
|
10.02+0.03
|
|
20.0
|
31.14
|
19.86+0.04
|
|
40.0
|
62.97
|
40.16+0.06
|
*Values are mean of three determinations with standard deviation
(+)
Table 5: Pulse polarographic determination of Dihydralizine
sulphate
|
Amount taken, μg
|
DPP
|
|
Mean peakcurrent, ip, μA
|
Amount found*, μg
|
|
2
|
2.72
|
2.51+0.02
|
|
5.0
|
5.40
|
4.98+0.03
|
|
10.0
|
10.82
|
9.98+0.07
|
|
20.0
|
21.48
|
19.82+0.08
|
|
40.0
|
43.06
|
39.72+0.08
|
The method has been extended to the analysis of
commercial drugs formulated from them. The recoveries have been in the ranges
98.0-100.5; 97.2-99.8 and 98.7-100.2 with RSD's in the ranges 0.2-0.5, 0.2-0.4
and 0.2-0.4; respectively (Tables 6,7 and 8).
Figure
7: Typical differential pulse polarogram of Dihydralazine sulphate as
dihydralazinium-dithiocarbazate in pyridinium perchlorate. 1-pyridinium
perchlorate; 2- dihydralazinium-dithiocarbazate.
CONCLUSION:
The great advantages of non aqueous pulse polarography
being more sensitive allowing analysis even at larger
dilutions (upto 10-7 mol L-1) and with smaller volumes of
sample solutions (upto 0.1 mL) without any interference from inert carriers commonly
present in formulations has
been applied successfully to the analysis of isoniazid, Hydralazine hydrochloride and Dihydralazine
sulphate drugs.
CONFLICT OF INTEREST:
The
authors declare that there is no conflict of interests regarding the
publication of this article.
ACKNOWLEDGEMENT:
The author is thankful to the management of
Institute of Engineering and Technology Bhaddal, Ropar for extending support to
complete this research work.
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