Professional Documents
Culture Documents
Abstract: A simple, fast, accurate and precise reverse phase high performance liquid
chromatographic method has been developed for the estimation of Spironolactone in oral
suspension. The chromatographic separation was achieved on Zorbax RX-C18 (150 x
4.6) mm; 5 m column with an isocratic mixture of methanol: water (60:40). The
injection volume was kept at 20l with mobile phase at a flow rate of 1 ml/min. The
wavelength of detection was kept at 240 nm with column temperature at 25 C. The
retention times for Spironolactone, Methyl paraben and Propyl paraben was found to be
6.610.04 min, 2.250.04 min and 4.670.04 min, respectively. Methyl paraben and
Propyl paraben were major excipients of the oral solution. The linearity was obtained in
the range of 250-600 g/ml, 50-120 g/ml and 5-12 g/ml with correlation coefficient
0.998, 0.999 and 0.999 for Spironolactone, Methyl paraben and Propyl paraben,
respectively. The proposed method was validated as per ICH guidelines and successfully
applied for the determination of investigated drugs in oral suspension.
Keywords: Spironolactone, metyl paraben, propyl paraben, oral suspension, RP-HPLC.
2196 J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3; 2196-2204.
Development Bapna et al.
INTRODUCTION
Materials: The formulation Spironolactone in oral suspension (label claim: Spironolactone 2 gm/200ml,
Methyl paraben 0.4 gm/200ml and Propyl paraben 0.04 gm/200ml), manufactured by Thames laboratory
ltd. was procured from analytical research laboratory, Ahmadabad. All the chemicals used were of
analytical grade and were purchased from MERCK Chem. Ltd., Mumbai.
2197 J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3; 2196-2204.
Development Bapna et al.
Instruments: Following instruments with given specification were used for estimation of SPIRO from
oral suspension, HPLC (Shimadzu) LC2010CHT with SPDM20A diode detector, LC solution software
was applied for data collecting and processing, pH meter (ELICO), digital balance (Sartorius-0.1
mg 205 gm) and FTIR Spectrophotometer (Brukeroptics).The chromatographic separation was achieved
on a Zorbax RX-C18 (150 x 4.6) mm; 5m as a stationary phase with isocratic elution. The mobile phase,
methanol: water (60:40) was pumped at a flow rate of 1 ml/min. The samples were analysed by a PDA
detector at 240 nm with the injection volume of 20 L.
METHOD DEVELOPMENT
Preparation of standard solution for Spironolactone: Accurately weighed 250 mg of standard SPIRO
was transferred to 50 ml volumetric flask and dissolved in 30 ml of diluent and shaken vigorously. The
volume was made up to the mark with diluent to give a solution containing 5000 g/ml of SPIRO. From
this 20 ml of solution was taken out and diluted up to 50 ml with diluent in a volumetric flask to give
solution of 2000 g/ml of SPIRO. Further, 2.5 ml of this solution was taken and diluted up to 10 ml with
diluent to give a working standard solution containing 500g/ml of SPIRO.
Preparation of standard solution for MP: Accurately weighed 100 mg of MP was transferred to a 50
ml volumetric flask and dissolved in 30 ml diluent. The flask was then shaken and volume was made up
to the mark with diluent to give a solution containing 2000 g/ml MP. Ten ml of this solution was taken
and diluted up to 50 ml with diluent in volumetric flask to give solution of 400 g/ml of MP. Further, 2.5
ml solution was taken and diluted up to 10 ml with diluent to give working standard solution containing
100 g/ml MP.
Preparation of sample solution: One ml of oral suspension was taken and diluted up to 20 ml with the
diluent. According to label claim 1 ml of suspension contains 10 mg SPIRO, 2 mg MP and 0.2 mg PP
therefore, after dilution 1 ml of suspension in 20 ml of diluent will contain 500 g/ml, 100 g/ml and 10
g/ml of SPIRO, MP and PP, respectively.
Calibration curve: Calibration curves constructed were linear over the concentration range of 250-600
g/ml, 50-120 g/ml and 5-12 g/ml for SPIRO, MP AND PP, respectively. Calibration curves were
prepared using ratio of analyte peak area to internal standard peak versus concentration of analytes. The
calibration curves are shown in Figure 1, 2 and 3.
2198 J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3; 2196-2204.
Development Bapna et al.
2199 J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3; 2196-2204.
Development Bapna et al.
METHOD VALIDATION
The proposed method was validated in accordance with ICH guidelines for accuracy, precision, LOD,
LOQ, linearity and percentage recovery.
Linearity: Linearity study was carried out for SPIRO, MP and PP at six different concentration levels.
Calibration curves constructed were linear over the concentration of 250-600 g/ml, 50-120 g/ml and 5-
12 g/ml for SPIRO, MP and PP, respectively. Evaluation of these drugs was performed with UV
detector at 240 nm and peak area was recorded for all the peaks. The correlation coefficient for SPIRO,
MP and PP were found to be 0.998, 0.999 and 0.999, respectively.
Limit of detection and limit of quantification: The LOD and LOQ of the developed method were
determined by injecting progressively low concentration of the standard solutions using the developed
HPLC method. The LOD for SPIRO, MP and PP were found to be 17.832 g/ml,1.335 g/ml and 0.5657
g/ml respectively. The LOQ for SPIRO, MP and PP were found to be 54.038 g/ml, 4.047 g/ml and
1.714 g/ml, respectively.
Accuracy: The accuracy of the method was assessed by recovery studies of SPIRO, MP and PP in
combined dosage form at three concentration levels. A fixed amount of pre-analyzed sample was taken
and standard drug was added at 50%, 100% and 200% levels. Each level was repeated for three times as
shown in Table-1. The percentage recoveries of SPIRO, MP and PP were 101.64%, 100.16% and 101.8%
which shows that there is no interference from excipients and the lower values of RSD of assay indicate
the method is accurate.
Table-2: Intra-day and Inter-day precision for the analysis of SPIRO, MP and PP.
2200 J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3; 2196-2204.
Development Bapna et al.
Precision: The precision for the developed method was determined in terms of intraday and inter-day
precision. For intraday precision evaluation, a standard solution of fixed concentration was injected at
various time intervals on a particular day and % RSD for SPIRO, MP and PP were found to be 0.64%,
0.82% and 0.62%, respectively (limit %RSD < 2.0%). In addition, the inter-day precision was studied by
injecting the same concentration of standard solution on consecutive days and the %RSD for SPIRO, MP
and PP were found to be 0.65%, 1.19% and 0.84%, respectively (limit %RSD < 2.0%). The results are
shown in Table-2.
Assay: Twenty microlitre of sample solution was injected and from the peak area of SPIRO, MP and PP
amount of each drug in sample was computed. The results of the assay (Table-3) undertaken, yielded
101.64%, 100.16% and 101.8% of SPIRO, MP and PP, respectively.
Force degradation studies: Whole stability indicating RP-HPLC assay method for simultaneous
determination of SPIRO, MP and PP were done using above developed RP-HPLC method. In order to
establish stability-indicating nature of the method, standards of drugs, drug product and diluent were
subjected to various stress conditions to conduct force degradation studies. Stress studies were carried out
under the conditions of acidic, basic, oxidative, thermal and UV exposure. Several trials with different
severity of each stressed condition were conducted, the results of which are shown in Table-4.
2201 J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3; 2196-2204.
Development Bapna et al.
RESULTS AND DISCUSSIONS
Estimation of SPIRO was carried out using optimized HPLC method. The method was linear in the range
of 250-600 g/ml, 50-120 g/ml and 5-12 g/ml for SPIRO, MP and PP, respectively. The linearity
curves for SPIRO, MP and PP are shown in Figure 1, Figure 2 and Figure 3. The samples were analysed
by a PDA detector at 240 nm with the injection volume of 20 L. The resultant peaks were good in shape
and resolution (Figure 4). The percentage recoveries of SPIRO, MP and PP were 101.64%, 100.16% and
101.8% which shows that there is no interference from excipients and the lower values of RSD of assay
indicate the method is accurate. The % RSD of SPIRO, MP and PP for intraday precision study were
found to be 0.64%, 0.82% and 0.62% respectively (limit %RSD< 2.0%). and % RSD of SPIRO, MP and
PP for inter-day precision study were found to be 0.65%, 1.19% and 0.84%, respectively.(limit %RSD <
2.0%).
Figure 4: Typical chromatogram of standard for Spironolactone, Methyl Paraben and Propyl Paraben.
The retention time of SPIRO, MP and PP was found to be 6.61min, 2.25min and 4.67min, respectively
with an asymmetry factor of 1.06 for SPIRO, 1.13 for MP and 1.01 for PP, which indicates efficient
performance of the column. The LOD for SPIRO, MP and PP were found to be 17.83259g/ml,
1.33575g/ml and 0.5657g/ml respectively. The LOQ for SPIRO, MP and PP were found to be
54.03816g/ml, 4.04773g/ml and 1.7141g/ml, respectively which indicate good sensitivity of the
proposed method. Assay studies of the proposed method indicate 101.64%, 100.16% and 101.8%
recovery for SPIRO, MP and PP, respectively. The results of the assay are shown in Table-4. No
interfering peaks were found in the chromatogram of the formulation within the run time indicating that
excipients used in formulations did not interfere with the estimation of the drug by the proposed HPLC
method.
2202 J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3; 2196-2204.
Development Bapna et al.
CONCLUSION
The developed HPLC method is simple, specific, accurate and precise for the simultaneous estimation of
SPIRO, MP and PP in oral liquid suspension. The developed method provides good resolution between
SPIRO, MP and PP. It was successfully validated in terms of linearity, accuracy, precision, LOD, LOQ
and recovery in accordance with ICH guidelines. Thus the described method is suitable for routine
analysis and quality control of pharmaceutical preparations containing these drugs in combinations.
REFERENCES
2203 J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3; 2196-2204.
Development Bapna et al.
spironolactone and torsemide in pharmaceutical dosage form International Journal
of Research in Ayurveda and Pharmacy. 2010,1,459-467.
13. M. Bhojani, Development and Validation of RP-HPLC Method for Simultaneous
Estimation of Furosemide and Spironolactone in their Combined Tablet Dosage
Form Journal of Pharmaceutical Sci. and Bioscientific Reseach. 2012, 2, 144-147.
14. L.R. Khanchandani. Development and validation of liquid chromatographic method
for simultaneous estimation of Spironolactone and Hydroflumethiazide in
pharmaceutical dosage form Novus International Journal of Chem. 2013, 2, 13-19.
15. G.S. Devika, Isocratic RP-HPLC Method for Simultaneous Estimation of
Spirinolactone and Hydrochlorthiazide in Oral Solid Dosage Form, Research Journal
of Pharmacy and Technology. 2012, 5, 1050-1053.
2204 J. Chem. Bio. Phy. Sci. Sec. B, May 2014 July 2014; Vol.4, No.3; 2196-2204.