INTRODUCTION:
Pioglitazone hydrochloride
(±)-5-{4-[2-(5-ethyl-2-pyridyl)ethoxy]benzyl}-2,4thiazolidinedione
hydrochloridsalt, is an oral anti-diabetic agent that has been shown to affect
abnormal glucose and lipid metabolism associated with insulin resistance by
enhancing insulin action on peripheral tissues in animal models)[1,2]. It is used in the treatment of type-II diabetes
(non-insulin-dependent diabetes mellitus, NIDDM also known as adult on set
diabetes).
Pioglitazone decreases insulin resistance in the
periphery and liver, resulting in increased insulin-dependent glucose disposal
and decreased hepatic glucose output., see Scheme 1.
Scheme 1: Chemical structure of pioglitazone
hydrochloride (PGZ).
Pioglitazone hydrochloride is an odorless white
crystalline powder that has a molecular formula of C19H20N2O3S•HCl
and a molecular weight of 392.90 g. It is soluble
in N,N-dimethylformamide, slightly soluble in anhydrous ethanol, very
slightly soluble in acetone and acetonitrile, practically insoluble in water,
and insoluble in ether[3,4].
Bromocresol green C21H14Br4O5S
(BCG), acts as a weak acid in solution. It can thus be in protonated or
deprotonated form, appearing yellow or blue, respectively, mol. mass 698.01 g [5], see scheme 2.
Scheme 2: Chemical structure of Bromocresol green (C21H14Br4O5S).
A simple, rapid and extractive
spectrophotometric method was developed for the determination of pioglitazone
hydrochloride in pure and pharmaceutical formulations. This method is based on
the formation of yellow ion-pair complex between the basic nitrogen of the drug
and bromocresol green (BCG) in phthalate buffer of pH 2.4. The formed complexes
were extracted with chloroform and measured at 419 nm)[6]. Various
spectrophotometric methods[7-19]
have been reported for the determination of pioglitazone hydrochloride in pure
as well as in dosage forms. Most spectrophotometric methods employ extraction
procedures. The complex extraction technique has some difficulties and
inaccuracies due to incomplete extraction or the formation of emulsions between
the hydrocarbon solvent and the basic compound-containing solution. In response
to the problems resulting from the extraction of the complex, it is better to
determine formed complex without extraction[20].
Also, none of the direct methods reported in the literature are based on the
formation of a complex between BCG and PGZ. Several analytical methods for the determination of pioglitazone hydrochloride have been reported including
high-performance liquid chromatography (HPLC) [21-25] and electrochemical
methods) [26-29].
In this study, a extraction-free
spectrophotometric method for determination of PGZ
through ion-pair complex formation with BCG was developed.
MATERIALS AND METHODS:
Instruments and apparatus:
Spectrophotometric measurements were made in Spectro
scan 80 DV UV-VIS spectrophotometry with 1 cm quartz cells. An ultrasonic
processor model Powersonic 405 was used to sonicate the sample solutions. The
diluter pipette model DIP-1 (Shimadzu), having 100 μL sample syringe and
five continuously adjustable pipettes covering a volume range from 10 to 1000
μL (model Piptman P, Gilson). Centrifuge (Centurion Scientific Ltd.,
Model: K2080- Manufactured in the United Kingdom) was used for the preparation
of the experimental solutions. Sartorius TE64 electronic balance was used for
weighing the samples.
Reagents:
Pioglitazone hydrochloride (99.37%) was supplied by Doshil
Group Company (India), (Mfg. 11/2015, Exp. 10/2020). Bromocresol green (97%) of
analytical grade and chloroform of extra pure were from Merck. All solvents and
reagents were analytical grade chemicals.
Stock standard solution of
bromocresol green (BCG) 1x10-2 mol.L-1:
Accurately weighed 179.9 mg of BCG was dissolved in
chloroform into a volumetric flask (25 mL) and diluted up to mark with
chloroform.
Stock standard solution of PGZ 1x10-3
mol.L-1:
This solution was prepared in two stages: the first
one (1x10-2 mol.L-1) by dissolving 98.85 mg of PGZ.HCl in
chloroform into a volumetric flask (25 mL) and the second 1x10-3
mol.L-1.
Working standard solutions of PGZ:
The stock solution was further diluted daily just
before the use to obtain working solutions of PGZ.HCl in the concentrations:
1.00, 2.00, 3.00, 5.0, 7.00, 10.00, 20.00, 30.00, 50.00, 70.00 and 100.00
μM (0.3929, 0.7858, 1.1787, 1.9645, 2.7503, 3.929, 7.858, 11.787, 19.645,
27.503 and 39.29 μg.mL-1 of PGZ.HCl) by transferring different
aliquots from stock standard solution: 10, 20, 30, 50, 70, 100, 200, 300, 500,
700 and 1000 μL into 10 mL volumetric flasks, then 0.10 and 0.5 mL from
stock standard solution of BCG (1x10-2 mol.L-1) for
concentrations 1.0–10.0 and 1.0-100.0 μM, respectively, were added, and
diluted to 10 mL with chloroform.
Sample preparation:
Commercial formulations (as a tablet) were used for
the analysis of PGZ. The pharmaceutical formulations subjected to the
analytical procedure were:
(1) Pioglit
tablets, Barakat pharmaceutical industries, Aleppo–SYRIA, each tablet
contains 15 and 30 mg of Pioglitazone hydrochloride(PGZ-HCl) (Mfg. 05/2014,
Exp. 05/2018 and Mfg. 03/2015, Exp. 03/2019, respectively).
(2) Actazone Asia
tablets, Asia pharmaceutical industries, Aleppo–Syria, each tablet contains
30 mg of PGZ.HCl (Mfg. 06/2015, Exp. 06/2018).
(3) Defast tablets,
Unipharma Pharmaceutical Industries. Damascus -Syria, each tablet contains 30mg
of PGZ.HCl (Mfg. 04/2015, Exp. 04/2018).
Stock solutions of pharmaceutical
formulations:
20 tablets of each studied pharmaceutical formulation
were weighed accurately, crushed to a fine powder and mixed well. An amount of
the powder equivalent to the weight of one tablet was solved in chloroform
using ultrasonic for 10 min, 20 mL of chloroform was added, filtered over a 100
mL flask and washed by the same solvent, then diluted to 100 mL with
chloroform. This solution contains the follows:150 and 300 μg.mL-1
of PGZ.HCl for all studied pharmaceutical formulations contain 15 and 30
mg/tab, respectively.
Working solutions of pharmaceuticals:
Five solutions were prepared daily by diluting 100 and
50 μL from a stock solution of pharmaceutical formulations for contents:
15 and 30mg/tab, respectively. Then adding 1 mL from a stock standard
solution 1x10-3 M of BCG and adjusting the volume up to 10 mL
with chloroform (these solutions contain 150 μg.mL-1 of PGZ.HCl; test solutions).
Procedure:
A solution (10 mL) containing an appropriate
concentration of PGZ.HCl (or working solutions of pharmaceuticals) with
appropriate amount of BCG in chloroform was ready for spectrophotometric
measurement at λmax 420 nm.
RESULTS AND DISCUSSION:
The effect of
solvent:
The effect of the solvents (acetone, acetonitrile,
dichloroethane, dichloromethane, chloroform and ethylacetate) on absorbance of
reagent (BCG), formed complex [PGZ]:[BCG] and the difference between
them. It was found that chloroform solvent was the best, see Figure 1.
Absorption Spectra:
UV-Vis spectra of PGZ, BCG and the formed
complex PGZ:BCG solutions in chloroform was obtained.
PGZ solutions do not absorb in the range 300-600 nm. BCG solutions have small
absorption at λmax
420nm (e≈435L.mol-1.cm-1 in chloroform). [PGZ]:[BCG] complex solutions have maximum
absorption at λmax 420 nm in chloroform, e for the complex was 14300
L.mol-1.cm-1, see Figure 2 as example.
Optimization of Variables:
The different experimental parameters
affecting the spectrophotometric determination of PGZ
through ion-pair complex [PGZ]:[BCG] formation with BCG in chloroform was
studied in order to determine the optimal conditions for the determination of PGZ.
Fig. 1: Absorbance of reagent (BCG), formed complex
[PGZ]:[BCG] and difference between them (CBCG 1x10-4
M, CComplex 5x10-5 M, Blank is solvent, ℓ =1 cm).
Fig.2. UV-Vis spectra in chloroform of: 1-
1x10-4 mol.L-1 of PGZ; 2-1.0x10-4 mol.L-1
of BCG; 3- 0.5x10-4 mol.L-1 ion-pair complex (0.5x10-4 mol.L-1 of
PGZ with 1x10-4 mol.L-1 of BCG); Blank is
1.0x10-4 μg.mL-1 of BCG, 4- 0.5x10-4
mol.L-1 ion-pair
complex (0.5x10-4 mol.L-1of
PGZ with 1.0x10-4 mol.L-1 of BCG ); Blank is
chloroform, ℓ =1 cm.
The effect of time and temperature:
The effect of time and temperature on the
complex [PGZ]:[BCG] formation was studied within the ranges 5-120 min and 15-60o
C. It was found that the formed complex wasn't affected by time after 60
min and temperature at those ranges.
The effect of BCG concentration:
The effect of BCG concentration on complex
[PGZ]:[BCG] formation was investigated. It was observed that the absorbance of the formed complex increased coinciding
with increasing the ratio of CBCG:CPGZ until the ratio (1:1), then slowly increased until the
absorbance became a quasi-static at ratio more than 10.
Stoichiometric Relationship:
The molar ratio method:
The composition of PGZ:BCG
complex were determined by the
molar ratio method and Job's method of continuous variation [30]. The stoichiometry of PGZ:BCG complex was studied by molar ratio method
according to following equation: Amax= f ([BCG]/[PGZ]) at λmax 420nm in chloroform. It confirmed
that the binding ratio of PGZ:BCG complexes are equal to (1:1); where
the concentration of PGZ was constant (50 µM) and the
concentrations of BCG changed from 0 to 300 µM, see Figure 3. The formation constant of the ion pair
complex [PGZ]:[BCG] is 4.2x107 in chloroform.
Fig.3. Molar ratio method to calculate binding ratio of
PGZ:BCG complex at λ=420 nm in chloroform (
[PGZ]= 50 µM, blank is chloroform, ℓ =1 cm).
The Job's method:
Continuous variation was utilized to check
the composition of PGZ:BCG complex at
λmax 420 nm in
chloroform. The absorbance of the complex in used solvent were plotted against
the mole fraction [BCG]/([PGZ]+[BCG]), where [PGZ]+[BCG]=100 µM.
The plot reached maximum value at a mole
fraction of 0.5, see Figure 4. This indicated complex formation (PGZ:BCG) in the ratio of (1:1). The formation constant of the ion- pair complex [PGZ]:[BCG] is 2.35x107.
Fig.4. Job's method of continuous variation
to calculate binding
ratio of PGZ:BCG complex at λ 420 nm in chloroform [PGZ]+[BCG]=100µM, blank is chloroform, ℓ =1 cm).
Mechanism of reaction:
Anionic dyes such as BCG form ion-pair
complexes with the positively charged nitrogen-containing molecule. The colour
of such dyes is due to the opening of lactoid ring and subsequent formation of
quinoid group (deprotonated). Pioglitazone
hydrochloride (C19H20N2O3S.HCl ) dissolved in chloroform and forms yellow ion-pair complex PGZ:BCG with dye. Each drug-dye complex with two oppositely
charged ions (positive on the drug and negative on the dye) behaves as a single
unit held together by an electrostatic binding [31-34]. The suggested mechanism
of PGZ-BCG ion-pair complex formation is shown in
Scheme 3.
Applications:
The developed spectrophotometric method was
applied to determine PGZ in
some pharmaceutical preparations through
complex formation
by BCG in chloroform according
to the optimal conditions. The results of
quantitative analysis for PGZ in pharmaceutical preparations were summarized in Table 3. The proposed method was simple, direct, specific and successfully
applied to the determination of PGZ in pharmaceuticals without any interference
from excipients. Average recovery ranged between 99.1 to 101.0%. The results obtained by this method agree well with
the contents stated on the labels and were validated by RP-HPLC method [21].
METHOD VALIDATION:
The developed method for simultaneous
estimation of PGZ has been validated in accordance with the International
Conference on Harmonization guidelines (ICH) [35].
Selectivity:
Selectivity test determines the effect of
excipients on the assay result. To determine the selectivity of the method,
standard solution of PGZ, commercial product solution and blank solutions were
analyzed. The results of the tests proved that the components other than the
drug did not produce any interfere.
Linearity:
Several aliquots of standard stock solution
of PGZ were taken in different 10 mL volumetric flask and diluted up to the
mark with chloroform such that their final concentrations were 0.3929–39.29
μg.mL-1 for PGZ-HCl. Absorbance was plotted against the
corresponding concentrations to obtain the calibration graph, see Figure 5 and
Table 3. Linearity equations obtained were y = 0.0365x + 0.0034 for the range
0.3929–39.29 μg.mL-1 (R2=0.9995).
Precision and Accuracy:
The precision and accuracy of proposed method was checked by recovery
study by addition of standard drug solution to pre-analyzed sample solution at
three different concentration levels (80%, 100% and 120%) within the range of
linearity for PGZ. The basic concentration level of sample
solution selected for spiking of the PGZ.HCl standard
solution was 11.787μg.mL-1. The proposed method was validated statistically and through recovery
studies, and was successfully applied for the determination of PGZ.HCl in pure
and dosage forms with percent recoveries ranged from 99.5% to 102.0%, see Table
4.
Table 4: Results of recovery studies (n=5).
|
Level
|
% Recovery
|
|
80%
|
99.5
|
|
100%
|
102.0
|
|
120%
|
101.6
|
Repeatability:
The repeatability was evaluated by performing 10
repeat measurements for 11.787μg.mL-1 of PGZ.HCl
using the studied spectrophotometric method under the optimum conditions.
The found amount of PGZ (
± SD) 11.976±0.3230μg.mL-1
and the percentage recovery was found to be 101.6 ± 3.25 with RSD of
0.032. These values indicate that the proposed method has high repeatability
for of PGZ.HCl analysis.
Sensitivity (LOD and LOQ):
The sensitivity of the method was evaluated
by determining the LOD and LOQ. The values of LOD and LOQ for of PGZ.HCl are 0.055 and 0.170 μg.mL-1,
respectively.
Robustness:
The robustness of the method adopted is
demonstrated by the constancy of the absorbance with the deliberated minor
change in the experimental parameters such as the change in the concentration
of excipients, CBCG:CPGZ (10±5%), temperature (20±5oC),
stability (10±5% h) and reaction time (60±2 min), see Table 5 which indicates the robustness of the
proposed method. The
absorbance was measured and assay was calculated for five times.
Specificity:
The specificity of the method was
ascertained by analyzing standard of PGZ in presence of
excipients. There was no interference from most of the common excipients.
Table 5: Robustness of the proposed spectrophotometric method.
|
Experimental parameter
variation
|
Average recovery (%)*
|
|
CPGZ.HCl
|
|
1.1787 µg.mL -1
|
27.503µg.mL -1
|
|
Temperature
15oC
25oC
|
99.2
99.4
|
100.0
100.3
|
|
Stability
9.5 h
10.5 h
|
99.7
100.6
|
99.8
100.5
|
|
Reaction time
58 min
62 min
|
99.7
100.4
|
99.8
100.4
|
* n=5.
The homogenization of tablets:
The homogenization of tablets in terms of
the weight and the amount of drug was studied. We found that the mean weight
and amount drug in the tablets was 0.2003 ± 0.0086 g (i.e. ±4.3%), 0.2155 ±
0.0078 g (i.e. ±3.6%) for Pioglit tablets (15 and 30 mg/tab), 0.2133 ± 0.0055g
(i.e. ±2.6%) for Actozone Asia tablets (30 mg/tab) and 0.1496 ± 0.0032 g (i.e.
±2.1%) for Defast tablets (30 mg/tab), respectively. While the mean amount drug
in the tablets was 14.86±0.609 mg/tab (i.e. ±4.1%) and 30.15±1.025
mg/tab (i.e. ±3.4%) for Pioglit tablets (15 and 30 mg/tab), 29.88±0.777
mg/tab (i.e. ±2.6%) for Actozone Asia tablets (30 mg/tab) and
30.31±0.636 mg/tab (i.e. ±2.1%) for Defast tablets (30
mg/tab), respectively; which shows that homogeneity of tablets is acceptable.
Interferences:
Metformin HCL up to 1000 mg and 2 mg
glimepiride with 15 mg of pioglitazone HCL does not interfere, while 5 mg of
glibenclamide with 15 mg pioglitazone HCL interfere (about 4%) and 60 mg of
gliclazide with 15 mg Pioglitazone HCl interfere ( about 35%).
CONCLUSION:
The developed spectrophotometric method is
simple, direct (extraction-free) and cost-effective for the determination of
PGZ in pure and tablet dosage
forms. This method is based on
formation of ion-pair complex between PGZ and BCG in chloroform ([PGZ]:[BCG].
Beer’s law in the optimum experimental conditions using [PGZ]:[BCG] complex is
valid within a concentration range of 0.3929-39.29 μg.mL-1. The
developed method is applied for the determination of PGZ in pure and its
commercial tablets without any interference from excipients with assay of 99.1
to 101.0% and average recovery of 99.5 to102.0%.
CONFLICT OF INTERESTS:
The authors have declared that no conflict
of interests exists.
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