Development and Validation of Stability Indicating HPLC

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Research Article
DOI:10.13179/canchemtrans.2015.03.01.0151
Development and Validation of Stability Indicating HPLC
Method for Quality Control of Pioglitazone Hydrochloride
Safwan Ashour1*, Amir Alhaj Sakur2 and Manar Kudemati1
1
2
*
Department of Chemistry, Faculty of Sciences, University of Aleppo, Aleppo, Syria and
Departmentof Analytical and Food Chemistry, Faculty of Pharmacy, University of Aleppo, Aleppo, Syria
Corresponding Author, E-mail: [email protected]
Received: September 28, 2014 Revised: November 17, 2014 Accepted: November 18, 2014 Published: November 21, 2014
Abstract: A simple, rapid and sensitive RP-HPLC method for the quantification of pioglitazone
hydrochloride in bulk drug and tablet formulation was developed and validated. Chlordiazepoxide was
used as internal standard. The separation was achieved on a Nucleodur 100-5 C18 column (250 mm× 4.6
mm i.d., 5 m particle size). The mobile phase consisted of methanol-0.07 M formic acid (57:43, v/v) at a
flow rate of 0.9 mL/min and detection was performed at 266 nm using photodiode array (PDA) detector.
The drug was subjected to various ICH prescribed stress conditions including hydrolysis (acid and
alkaline), oxidation, photolysis and thermal degradation. Degradation in acid, base and peroxide was
found in range 36-45%. The proposed method was validated with respect to specificity, linearity,
accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), stability, and robustness as per
ICH guideline. The developed method was found to be successively applied for the quality control of
pioglitazone hydrochloride in bulk drug and tablets as well as the stability indicating studies.
Keywords: RP-HPLC; pioglitazone hydrochloride; stability indicating; analysis; validation.
1. INTRODUCTION
Pioglitazone hydrochloride (Figure 1), (±)-5-[p-[2-(5-Ethyl-2-pyridyl)ethoxy]benzyl]-2,4thiazolidinedione monohydrochloride, is an oral antidiabetic agent used in the treatment of type 2 diabetes
mellitus (also known as non‐insulin‐dependent diabetes mellitus [1,2] (NIDDM) or adult‐onset diabetes).
Pioglitazone decreases insulin resistance in the periphery and liver, resulting in increased insulindependent glucose disposal and decreased hepatic glucose output. Few methods for the determination of
pioglitazone hydrochloride were reported including voltammetry in tablets and biological fluids [3],
potentiometry in tablets [4], extractive spectrophotometry in raw material and tablets [5], HPLC in
pharmaceutical formulations [6-11] and in biological fluids [11-14]. Pioglitazone hydrochloride has been
determined in combination with other drugs using UV-spectrophotometry [15-17], HPLC [15,18-21] in
combined dosage forms, HPLC in formulations and human serum [22-25] and LC-MS methods in human
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plasma [26-28]. United States Pharmacopoeia (USP) [29] employ HPLC method for the assay of
pioglitazone hydrochloride in tablets. Further, the USP describes no environmentally friendly
chromatographic procedure since acetonitrile is one of the components of mobile phase. The literature
survey reveals that none is reported as a stability indicating assay method. Therefore, the aim of this study
is focus on the development and validation of a rapid, sensitive and accurate stability-indicating RPHPLC method for the determination of pioglitazone hydrochloride in bulk drug and tablet formulation
with a simple composition, low cost of mobile phase and lower solvent consumption leads to an
environmentally friendly chromatographic procedure. Chlordiazepoxide (Figure 1) was used as internal
standard, to improve the analytical performance and thus control undetermined changes in active
pharmaceutical ingredient concentration and instrument response fluctuations, and also to reduce the
problem of the many-fold dilution required in the classical batch procedures. Additionally, forced
degradation studies were achieved on the drug substance in accordance to the International Conference on
Harmonization (ICH) guidelines [30]. The method serves as an alternative to the methods described in
pharmacopoeias.
Figure 1. Chemical structure of pioglitazone HCl (A) and chlordiazepoxide (B).
2. EXPERIMENTAL
2.1. Materials
Pure drug samples of pioglitazone hydrochloride (PIO) and chlordiazepoxide (CLZ) were obtained
from Dr Reddys and Centaur Pharmaceuticals PVT (India), respectively. The HPLC-grade methanol and
water were purchased from Merck (Germany). Analytical reagent grade formic acid from Merck was used
to prepare the mobile phase. Tablets were purchased from Syrian market, containing pioglitazone
hydrochloride 15 or 30 mg per tablet.
2.2. HPLC system
The chromatographic system consisted of Hitachi (Japan) Model L-2000 equipped with a binary
pump (model L-2130, flow rate range of 0.000-9.999 mL/min), degasser and a column oven (model L2350, temperature range of 1-85 oC). All samples were injected using a Hitachi L-2200 autosampler
(injection volume range of 0.1-100 L). Elutions of all analytes were monitored at 266 nm by using a
Hitachi L-2455 absorbance detector (190-900 nm) containing a quartz flow cell (10mm path and 13L
volume). Each chromatogram was analyzed and integrated automatically using the Ezchrom Elite Hitachi
Software.
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2.3. Chromatographic conditions
Separation was achieved on a reversed phase Nucleodur 100-5 C18 column (250 × 4.6 mm, 5 m
particle size,). The mobile phase was consisted of methanol/0.07 M formic acid (43/57, v/v) and was
pumped at a flow rate of 0.9 mL/min. The mobile phase was filtered through a nylon 0.45 m membrane
filter and degassed by ultrasonic agitation before use. The injection volume was 10 L. The system was
operated at ambient temperature.
2.4. Standard solutions
Standard stock solution of PIO (1.0 mg/mL) was prepared by direct weighing of standard substance
with subsequent dissolution in HPLC grade methanol. From this stock solution the working standard
solution was prepared by further diluting of stock solution using methanol. Standard solution of CLZ (1.0
mg/mL) was prepared by dissolving appropriate amount of the compound in methanol. These solutions
were stored in the dark at 2-8 °C and found to be stable for two weeks at least.
2.5. Assay procedure for dosage form
Twenty tablets containing PIO were weighed and finely powdered. Portions of the powder (each
equivalent to the weight of five tablets) were accurately weighed into 50 mL volumetric flasks and 30 mL
methanol was added. The volumetric flasks were sonicated for 15 min to effect complete dissolution of
the PIO, the solutions were then made up to volume with methanol. The sample solutions were filtered
through 0.45 m nylon filter. The aliquot portions of the filtrate were further diluted to get final
concentration of 200 g/mL of PIO in the presence of 50 g/mL of internal standard. Finally, 10 L of
each diluted sample was injected into the column and chromatogram was recorded for the same. Peak
area ratios of PIO to that of CLZ were then measured for the determination of PIO concentrations in the
samples and then calculated using peak data and standard curves.
2.6. Forced degradation studies
To evaluate the stability indicating properties of the developed HPLC method, forced degradation
studies of PIO were executed in accordance with ICH guidelines as indicated below. Drug at a
concentration of 0.2 mg/mL was used in all degradation studies. After degradation all solutions were
diluted with methanol to yield starting concentration of 200 g/mL, filtered and then chromatographed
along with a non-stressed sample.
2.6.1. Hydrolytic degradation study
Sample solutions were treated separately with 0.1 N HCl and 0.1 N NaOH at 75 °C for 1 h (in a
heat block). The solutions were cooled and then neutralized as desirable (0.1 N NaOH or 0.1 N HCl).
2.6.2. Oxidative degradation study
Sample solutions were treated with solution of 3.0% (v/v) H2O2 at 75 oC for 1 h and cooled.
2.6.3. Thermal degradation study
PIO powder was exposed to heat at 100 °C for 24 h in a convection oven, and then cooled. An
amount of the powder was weighed and diluted as previously described.
2.6.4. Photo-degradation study
Active pharmaceutical ingredient (API) powder of PIO was prepared and exposed to UV-light up
to 24 h. Approximately 100 mg of powder was spread on a glass dish in a layer that was less than 2 mm in
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thickness. Under UV-light, all samples were placed in a photo-stability chamber. The UV radiation is at
254 nm. All stressed samples were taken out periodically and prepared as previously described.
2.7. Method Validation
2.7.1. Linearity
A series of working standard drug solutions equivalent to 0.99-701.00 g/mL were prepared by
diluting the stock standard solution with the methanol. In each sample 0.5 mL of CLZ was added (50
g/mL in the final volume). To construct the calibration curve five replicates (10 L) of each standard
solution were injected immediately after preparation into the column and the peak area of the
chromatograms were measured. Then, the mean peak area ratio of PIO to that of the internal standard was
plotted against the corresponding concentration to obtain the calibration graph.
2.7.2. LOD and LOQ
The minimum level at which the investigated compounds can be reliably detected (limit of
detection, LOD) and quantified (limit of quantitation, LOQ) were determined experimentally. LOD was
expressed as the concentration of compound that generated a response to three times of the signal to-noise
(S/N) ratio, and LOQ was 10 times of the S/N ratio [31]. The LOD and LOQ parameters were determined
from regression equations of PIO; LOD(k=3)=k×Sa/b, LOQ(k=10)=k×Sa/b (where b is the slope of the
calibration curve and Sa is the standard deviation of the intercept).
2.7.3. Precision and accuracy
Intra and inter-day precision of the method were determined by performing replicate (n = 5)
analyses of standards and samples. Intra-day assay variation was evaluated by injecting these samples in
the same day. Inter-day assay variation was evaluated by injecting these samples on 3 different days from
1 to 15 after preparation. Recovery study was performed in view to justify accuracy of the proposed
method.
2.7.4. Specificity
The specificity of the method was established through study of resolution factor of drug peaks from
nearest resolving peak and also among all other peaks. Peak purity of PIO was assessed to evaluate the
specificity of the method. Specificity was also studied by performing the forced degradation study using
acid and alkali hydrolysis, chemical oxidation, dry heat and photo degradation studies.
2.7.5. Robustness
Robustness of HPLC method was determined by deliberately varying certain parameters like flow
rate, percentage of organic solvent in mobile phase and pH of mobile phase. For all changes in conditions
the samples were analysed in triplicate. When the effect altering one set of conditions was tested, the
other conditions were held constant at optimum values.
2.7.6. System suitability
The system suitability test was performed to confirm that the LC system to be used was suitable for
intended application. A standard solution containing 200 g/mL of PIO in the presence of 50 g/mL of
internal standard was injected seven times. The parameters peak area, retention time, capacity factor,
selectivity, resolution, theoretical plates, tailing factor (peak symmetry) and % RSD were determined.
3. RESULTS AND DISCUSSION
3.1. Method development and optimization of chromatographic conditions
When acetonitrile and water was used drug peak merged with solvent front. When methanol and
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water was used as mobile phase drug eluted late and had broadening. So buffer was used. When 65:35
ratio of methanol: formic acid 0.07 M was tried drug peak was near to solvent peak. An increase in the
percentage of methanol decreases the retention of PIO and the internal standard, CLZ. Increasing
methanol percentage to more than 70% PIO peak is eluted with the solvent front, while at methanol
percentage lower than 50% the elution of CLZ peak is seriously delayed. The optimum methanol
percentage was found to be 57%. So ratio of 57:43 methanol: formic acid 0.07 M was gave satisfactory
result (Figure 2).
Nucledur100-5 C18 column gave the most suitable resolution between PIO and CLZ peaks (>4)
according to the pharmacopeial requirement while the other columns (Nucleodur C8, Hichrom 5 C8,
Nucleodur C18, ODS Hypersil C18) cause the peaks of the PIO and CLZ either to be overlapped or to
have unsuitable resolution (<4).
The use of isocratic elution was proven to be short retention time for the PIO peak and helped in
the separation of PIO, CLZ and degradation products. Figure 2 shows a typical chromatogram obtained
by the proposed RP-HPLC method, demonstrating the resolution of the symmetrical peaks corresponding
to PIO and CLZ with a flow rate of 0.9 mL/min. The retention time of PIO and CLZ was about 3.66, and
4.92 min, respectively. The retention time observed allows a fast determination of the drug, which is
suitable for QC laboratories. The optimum wavelength for detection was at 266 nm, at which the best
detector responses for all substances were obtained.
Figure 2. A typical chromatogram of a mixture of PIO (200 g/mL) and CLZ (50 g/mL) under optimal
conditions.
3.2. Accelerated degradation
The results of the forced degradation study are given in Table 1. PIO was found to be sensitive to
acid hydrolysis. About 40% degradation of PIO was found when heating in 0.1 N HCl at 75 oC for 1 h. A
chromatogram of the acid degraded standard solution is presented in Figure 3a. In alkali condition in 0.1
N NaOH at 75 oC for 1 h, about 45% of drug was degraded and produced a very minor unknown
degradation product at 7.3 min as shown in Figure 3b. Upon heating the solution in H 2O2 3% at 75 oC for
1 h, PIO yielded a minor degradation product at 2.76 min, whereas the degradation of PIO was at 3.79
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min with an assay of 63.39% (Figure 3c). PIO was found to be stable to the effect of light and
temperature. When the drug powder was exposed to UV light for one day and to dry heat at 100 oC for
one day, no decomposition of the drug was observed (Figure 3d,e).
Table 1. Force degradation of PIO by stability indicating RP-HPLC method.
Degradation mode
Control
Acid hydrolysis
Alkaline hydrolysis
Oxidation
Dry heat
Photolysis
Conditions
None
0.1 N HCl, 75 oC, 60 min
0.1 N NaOH, 75 oC, 60 min
3% H2O2, 75 oC, 60 min
100 oC, 24 hr
UV light at 254 nm, 24 hr
Retention time, min
3.733
3.640
3.833
3.787
3.727
3.713
% Assay of active ingredient
101.05
58.04
55.66
63.39
101.01
101.00
Table 2. System Suitability Parameters.
Parameters
Capacity factor (k')
Selectivity (
Resolution (Rs)
Number of theoretical plates (N)
Tailing factor (T)
% RSD for seven injections
PIO
2.49
–
–
4478
1.12
0.94
CLZ
3.66
1.47
4.10
4847
1.14
0.08
Preferable levels
2 – 10
1.0 – 2.0
> 1.5
> 2500
< 1.5
3.3. Method validation
The method was validated according to ICH guidelines. The following validation characteristics
were addressed:
3.3.1. System suitability
The system suitability requirements for PIO in the presence of CLZ was a % RSD for peak area
less than 0.94, a peak tailing factor less than 1.14 and Rs greater than 4.0 between adjacent peaks for the
analyte. This method met these requirements. The results are shown in Table 2.
3.3.2. Specificity
The specificity of the method was established through study of resolution factor of drug peaks from
nearest resolving peak and also among all other peaks. The specificity of the chromatographic method
was determined to ensure separation of PIO and CLZ as illustrated in Figure 2 where complete separation
of PIO was noticed. The HPLC chromatogram recorded for the analyte in tablet (Figure 4) showed almost
no peaks within a retention time range of 10 min. The figure show that PIO is clearly separated and the
peak of analyte was pure and excipients in the formulation did not interfere the analyte.
3.3.3. Linearity and limits of detection and quantification
A linear calibration graph was obtained with correlation coefficient of the regression equation [32]
greater than 0.999 in all cases and results are summarized in Table 3. Calibration plot was linear for 0.99701.00 µg/mL for PIO. LOD and LOQ were 0.16 and 0.54 g/mL, respectively, showed good sensitivity
of the proposed method.
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Table 3. Calibration data for the estimation of PIO by HPLC.
Parameters
Optimum concentration range (µg/mL)
Regression equation*
Correlation coefficient (r2)
Standard deviation of slope
Standard deviation of intercept
Regression equation**
Correlation coefficient (r2)
Standard deviation of slope
Standard deviation of intercept
Limit of quantification, LOQ (µg/mL)
Limit of detection, LOD (µg/mL)
PIO
0.99–701.00
APIO = 0.0464CPIO + 0.0074
0.9999
0.001
0.002
RPIO/CLZ = 0.0049C PIO + 0.0009
0.9999
3.2×10-4
0.0002
0.54
0.16
*Regression equation for the peak area of PIO vs concentration of PIO in g/mL.
**Regression equation for the ratio of peak area of PIO to that of I.S. vs concentration of PIO in g/mL.
Figure 3. HPLC chromatograms obtained for PIO from degradation studies.
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3.3.4. Accuracy and precision
Accuracy was determined by calculating the recovery. The method was found to be accurate with
% recovery 100.03%-104.66% for PIO (Table 4).
Precision: a) Repeatability data are shown in Table 5. The % RSD is < 2 for PIO which indicate that the
method is precise. b) Variation of results within the same day (intra-day), variation of results between
days (inter-day) was analyzed. For intra-day (n=5) % RSD was found to be 0.10–1.13% and % RSD for
inter-day (n=5) was 0.13-1.43%. The % RSD is < 2 which indicate that the method is precise (Table 4).
3.3.5. Robustness
The robustness was investigated by achieving deliberate changes in concentration of formic acid by
± 0.02 M, flow rate by ± 0.1 mL/min and change in methanol composition of mobile phase by ±2%.
Robustness of the method was carried out in triplicate at a concentration of 200 g/mL. The system
suitability parameters remained unaffected over deliberate small changes in the chromatographic system,
illustrating that the method was robust over an acceptable working range of its HPLC operational
parameters (Table 5).
Table 4. Accuracy and precision of within and between run analysis for determination of PIO by HPLC.
Concentration (g/mL)
0.99
5.00
10.00
50.00
100.00
200.00
400.00
600.00
701.00
Intra-day (n=5)
Mean
RSD
(%)
(g/mL)
1.03
1.13
5.05
1.03
10.28
1.07
50.25
1.01
101.50
0.99
202.19
0.94
402.35
0.71
606.55
0.19
710.29
0.10
Recovery
(%)
104.66
101.18
102.87
100.50
101.50
101.09
100.59
101.09
101.32
Inter-day (n=5)
Mean
RSD
(%)
(g/mL)
1.00
1.43
5.07
1.35
10.19
1.31
50.60
1.06
101.60
1.02
202.11
0.95
401.46
0.89
600.16
0.27
701.38
0.13
Recovery
(%)
101.01
101.40
101.90
101.20
101.60
101.05
100.36
100.03
100.19
Table 5. Results of robustness study.
Factor
Formic acid, M
Flow rate, mL/min
% of methanol
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0.05
0.09
0.8
1.0
55
59
PIO
% Mean assay (n = 3)
103.41
102.96
104.26
91.80
102.40
105.76
% RSD of results
1.94
0.62
0.60
0.91
0.69
1.42
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250
250
4.500
3.333
300
200
200
150
150
100
100
50
mAU
mAU
Ca
50
0
0
0
1
2
3
4
5
6
7
8
9
10
Minutes
Figure 4. A typical chromatogram of PIO (200 g/mL) and the internal standard CLZ (50 g/mL) in the
mobile phase, prepared from Pioglet tablets under the optimal conditions.
3.4. Method application
The proposed, developed and validated method was successfully applied to analysis of PIO in their
marketed formulation (Figure 4). There was no interference of excipients commonly found in tablets as
described in specificity studies. Student’s t-test was used for statistical analysis of the data and statistical
significance was defined at the level of P<0.05. The results obtained with the proposed method were
compared with the official method [29] and have been shown in Table 6. Good agreement with results
obtained by the official method was observed. The proposed method is simple, rapid, accurate, highly
sensitive and suitable for the routine quality control.
Table 6. Determination of PIO in tablets by the proposed and official methods.
Formulation
Actazone c (15 mg/tablet)
Pioglit d (15 mg/tablet)
Defast e (30 mg/tablet)
Recovery % a ± SD
Proposed method
100.480.44
100.440.34
101.110.79
Official method
99.30.47
100.70.54
103.30.86
t-value b
F-value b
2.18
2.62
2.08
1.17
2.55
1.20
a. Five independent analyses. b. Theoretical values for t and F-test at five degree of freedom and 95% confidence limit are t
=2.776 and F=6.26. c. Supplied by Asia, d. supplied by BPI and e. supplied by Unipharma, Syria.
4. CONCLUSION
This developed and validated method for analysis of PIO in pharmaceutical preparations is very
rapid, accurate, and precise. The method was successfully applied for determination of PIO in its
pharmaceutical tablet formulation with limit of detection of 0.16 g/mL. Moreover it has advantages of
short run time and the possibility of analysis of a large number of samples, both of which significantly
reduce the analysis time per sample. Hence this method can be conveniently used for routine quality
control analysis of PIO in its pharmaceutical formulation.
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The authors declare no conflict of interest
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