International Journal of Advances in Pharmaceutical Analysis
`IJAPA Vol. 4 Issue 2 (2014) 47-52
`Journal Home Page http://www.ijapa.ssjournals.com
`
`
`
`Stress degradation of Lisinopril as per ICH Guidelines &
`Characterisation
`
`S.K. Tuse*, A.R. Vadgaonkar, D.S. Musmade and V.S. Kasture
`
`
`Quality Assurance Department, Sanjivani College of Pharmaceutical Education &
`Research, Kopargaon, Maharashtra, India.
`Abstract
`Lisinopril an antihypertensive drug was subjected to stress degradation, since the drug is
`photosensitive undergo hydrolysis and oxidized in presence of oxygen. Hence the objective of the study was to
`stress degrade Lisinopril & to find out the pathway for stress degradation of Lisinopril. Stress testing methods
`are screening methods to be used to understand the degradation chemistry of a drug. Lisinopril was subjected
`to stress degradation under different conditions recommended by International Conference on Harmonization
`(ICH). The chromatographic separation of Lisinopril & its degradation products was done on C 18 column &
`mobile phase was mixture of Methanol & Water in ratio 80:20, pH 3.5 adjusted with orthophosphoric acid at a
`flow rate of 1ml/min using UV detector with λmax 220nm. The quantification & characterizations of degraded
`products were carried out by UV, IR spectroscopy & HPLC. The mechanism of degradation was confirmed by
`GC-MS fragmentation pattern.
`Keywords: Stress degradation, Lisinopril, ICH guidelines
`
`1. Introduction
`(2S)-1-[(2S)-6-amino-
`(LIS),
`Lisinopril,
`2[[(1S)-1-carboxy-3-phenylpropyl]
`amino]-
`hexanoyl]-pirrole-2-carboxylic acid1 used
`in
`the
`treatment of essential hypertension, symptomatic &
`asymptomatic left ventricular systolic dysfunction,
`post-myocardial infarction, renal failure & diabetic
`nephropathy. The analysis of Lisinopril was reported
`by RP-HPLC method in bulk & tablet dosage form. A
`novel RP-HPLC method was developed
`for
`simultaneous estimation of Lisinopril in combination
`for bulk & tablet dosage form2. The pharmaceutical
`products are prone to undergo degradation in various
`physical & chemical conditions & yield impurities
`which adversely affect the performance of drug
`substance. Hence, it has been mandated by regulatory
`agencies of various countries to submit the stability
`indicating data of the drug substance & the drug
`product before the approval for commercialization of
`products. Hence it is necessary to develop stability
`indicating method for analysis of drug substance &
`their
`impurities. There
`is no reported stability
`indicating analytical method for analysis of Lisinopril
`& its degradation products in bulk. The present work
`aimed at the stress degradation study of Lisinopril in
`bulk & establishment of structures of degraded
`product by sophisticated instrumental methods like
`UV, IR, RP-HPLC and GC-MS3.
`
`8400S. The high pressure liquid chromatographic
`(HPLC) system used was of model SHIMADZU
`8400S Prominence SPD-20Am Gradient System
`equipped with Hamilton injector & UV visible
`detector. A Phenomex HPLC column C18 reverse
`phase, 5µm, (50×4.6mm) was used. The GC- MS
`instrument of model- Accu TOF GCV, model no-
`7890 with FID detector, head space injector &
`combipal autosamlper & mass range 10-2000amu,
`mass resolution 6000 was used for analysis.
`2.2 Materials: Lisinopril bulk was obtained as a gift
`sample from Mylan Laboratories, Aurangabad, India.
`Methanol & water HPLC grade obtained from Fischer
`Scientific, Mumbai. Orthophosphoric acid (Sd fine
`chemicals).
`2.3 Methods
`2.3.1 Selection of Wavelength: The wavelength of
`maximum absorption for Lisinopril was recorded by
`UV spectrophotometer (figure 1).
`Fig no.1 UV spectrum for Lisinopril
`
`
`
`Structure of Lisinopril4
`2. Materials and Methods
`2.1 Instrumentation: The UV spectrophotometer
`was used of model SHIMADZU UV 1650-PC. IR
`spectrophotometer was used of model SHIMADZU
`
`
`2.3.2 Preparation of mobile phase: The mobile
`phase was prepared in the ratio 80:20 v/v (methanol:
`water). The pH was adjusted
`to 3.5 with
`orthophosphoric acid, filtered & degassed with
`sonication for 10 mins.
`
`Corresponding Author*: snehaltuse@gmail.com 47
`
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`2.4.6. Characterization of degraded product by
`GC-MS study: The fragmentation pattern was used
`for mechanism of degradation by various degraded
`process. Fig no.10, 12, 14, 16 respectively.
`
`3. Results and Discussion
`3.1 Method development
`3.1.1. UV Method development: Water was used as
`a solvent for recording UV spectrum. The λ max
`selected was 205 nm. The UV method was developed
`& validated as ICH guidelines. Table no. 1
`Table No.1 Summary for UV Method validation of
`Lisinopril
`Parameter
`
`Research Article Tuse et al /2014
`
`2.3.3. Preparation of Standard solution: UV
`spectrum was recorded using spectroscopic grade
`water as solvent5. For HPLC analysis, a standard
`stock solution was prepared by dissolving 100 mg of
`Lisinopril in 100 ml of mobile phase. 1 ml of above
`solution was diluted to 100 ml with mobile phase to
`obtain a concentration of 10µg/ml & further it was
`diluted to 100ml with mobile phase to obtain a
`concentration of 1 µg/ml. Lisinopril chromatogram is
`given in fig 4.
`2.3.4. Preparation of calibration curve: For
`calibration by UV spectrophotometer, six standards
`were prepared having concentrations in the range of
`3-18µg/ml using water as a solvent. (Table 1 & fig 2).
`For HPLC analysis, six standards were used having
`concentrations in the range 0.6-1.6 µg/ml diluting
`with mobile phase. (Table 2 & Fig 5)
`2.3.5. Recording IR spectra: The IR spectrum of
`Lisinopril was recorded using kBr pellet technique.6
`(fig no.3)
`2.3.6. System suitability Tests: System suitability
`was verified by injecting working standard of 1µg/ml.
`Various parameters such as HETP, number of
`theoretical plates, tailing factor & asymmetry were
`recorded. (Table 3)
`2.4. Validation parameters as per ICH guidelines:
`The UV & HPLC methods were validated in terms of
`precision, LOD, LOQ, linearity, range, ruggedness &
`robustness7.
`2.4.1. Precision: The precision of proposed methods
`were evaluated by carrying out six independent test
`values. Intraday & Interday precision were carried
`out.
`2.4.2. Limit of Detection & Limit of Quantitation:
`The
`limit of detection (LOD) & the limit of
`quantitation (LOQ) for Lisinopril by UV & HPLC
`were reported from standard deviation of the response
`& the slope.
` LOD= σ/S × 3.3
` LOQ= σ/S × 10
` 2.4.3. Robustness: The robustness of the method
`was determined as measure of the analytical method
`capability to be unaffected by deliberate small change
`in method parameters. The changes made such as use
`of single beam instrument for double beam, variation
`
`Linearity
`Slope
`Intercept
`Correlation Coefficient
`Precision
`
`Intraday Precision
`
`Interday Precision
`
`Ruggedness
`
`Robustness
`
`S.D
`% RSD
`S.D
`% RSD
`S.D
`%R.S.D
`S.D
`% RSD
`S.D
`%RSD
`
`Observation
`3-30µg/ml
`0.0291
`0.0259
`0.988
`0.0084
`1.92
`0.0074
`1.60
`0.0072
`1.58
`0.0049
`1.20
`0.0076
`1.64
`0.95
`LOD
`2.88
`LOQ
`3.1.2. HPLC Method development: Several mobile
`phase compositions were tried to resolve the peaks of
`Lisinopril & its degradation products. The mobile
`phase containing methanol- water 80:20 (v/v) was
`optimized for analysis since it resolved the peaks of
`Lisinopril (RT = 2.28 ± 0.02) with resolution factor of
`5.6. The pH was adjusted to 3.5 with orthophosphoric
`acid. The system suitability parameter was stated in
`Table no.3 Quantification was achieved with UV
`detection at 205nm on the basis of peak area. A
`typical chromatogram was obtained. The HPLC
`method developed & validated as per ICH guidelines
`for quantitation of force degraded products10. Table
`no. 2
`Table No.2 Summary for method validation of
`Lisinopril by HLPC
`Parameters
`Observations
`0.6- 1.6 μg/ml
`270.97
`10.79
`0.995
`1.250
`0.45
`0.463
`0.16
`0.652
`
`in flow rate by
`
` 0.2 ml/min, variation in wavelength
`
` 2nm. At these changed conditions the standard
`by
`solutions were analyzed. S.D & % R.S.D were
`calculated.
`2.4.4. Ruggedness: The ruggedness of the method
`was checked by changing analyst & expressed as S.D
`& % RSD.
`2.4.5. Force degradation study: To check the
`stability,
`the drug was forced degraded under
`acid/base hydrolytic, oxidative & Photolytic stress
`conditions as per ICH recommendation8.
`The drug was subjected to acid hydrolysis by
`using 0.1 N hydrochloric acid for 6hrs at 400C; base
`hydrolysis by using 0.1N sodium hydroxide solution
`for 6 hrs at 400C; oxidation by using 6% solution of
`hydrogen peroxide for 6 hrs & photolytic stress using
`sunlight for 72hrs9.
`
`
`
`Precision
`
`Intraday Precision
`
`Interday Precision
`
`Linearity
`Slope
`Intercept
`Correlation coefficient
`S.D
`%RSD
`S.D
`%RSD
`S.D
`%RSD
`S.D
`%RSD
`S.D
`%RSD
`
`Ruggedness
`
`Robustness
`
`LOD
`LOQ
`
`0.23
`0.430
`0.16
`0.726
`0.30
`0.015
`0.046
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`Research Article Tuse et al /2014
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`Table No.3 System suitability parameters for Lisinopril
`Retention time Tailing factor Asymmetry Theoretical plate Resolution
`2.28
`1.23
`1.89
`4426.65
`5.68
`
`
`Table No.4 Comparative data for % degradation by UV & HPLC
`Compounds
`Hydrolysis Acid Hydrolytic
`Hydrolytic
`Photolytic
`Base
`Base
`69.95%
`73.27%
`93.36%
`94.50%
`
`59.83%
`89.85%
`
`% Degradation by UV
`% Degradation by HPLC
`
`67.83%
`92.81%
`
`
`
`3.2 Force Degradation
` Forced degradation studies were carried out
`for Lisinopril in acid hydrolysis, alkaline hydrolysis,
`oxidation & photolytic stress11. The peaks of
`degradation components were well resolved &
`appeared at 2.3, 3.2, 2.8 & 2.9 respectively.fig no.6-9.
`The % degradation of Lisinopril was
`quantified by UV & HPLC methods the comparative
`data is presented in table no.4. Since HPLC is more
`specific & selective method the % degraded amount
`is more for HLPC as compared to UV method.
`3.3. Mechanism of degradation
`The structure of degraded products was
`confirmed by GC-MS study. The fragmentation patter
`was correlated with
`the structure of degraded
`products12.
`For acid hydrolysis degraded product formed
`Benzenebutanoic acid & the pathway of degradation
`was stated in fig no.11
`For base hydrolysis degraded product
`formed Benzene
`fragment &
`the pathway of
`degradation was stated in fig no.13
`For oxidation degraded product formed P-Toluene &
`the pathway of degradation was stated in fig no.15
`For photolysis degraded product formed & the
`pathway of degradation was stated in fig no.16.
`
`Fig no.2 Graph for Linearity of Lisinopril
`
`
`Fig no.4 HPLC chromatogram for Lisinopril
`
`
`Fig no.5 Graph for linearity for Lisinopril by
`HPLC
`
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`Fig no.6 HPLC chromatogram for acid hydrolytic
`degraded product
`
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`Fig no.7 HPLC chromatogram for base hydrolytic
`degraded product
`
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`Fig no.3 IR spectrum for Lisinopril
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`Research Article Tuse et al /2014
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`Fig no.8 HPLC chromatogram of oxidation
`degraded product
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`Fig no.11 Fragmentation pattern
`hydrolytic degraded product
`
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`for acid
`
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`Fig no.9 HPLC chromatogram of photolytic
`degraded product
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`Fig no.10 GC-MS spectra of degradation of
`Lisinopril by acid hydrolysis
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`Fig no.12 GC-MS spectra of base hydrolytic
`degraded product
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`Research Article Tuse et al /2014
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`Fig no.13 Fragmentation pattern for base
`hydrolytic degraded product
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`Fig no.14 GC-MS spectra oxidation degraded
`product
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`Fig no.15 Fragmentation pattern for oxidation
`degraded product
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`p- Toluene
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`Research Article Tuse et al /2014
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`
`Acknowledgement
`The authors are thankful to Mylan Lab,
`Aurangabad., India. Principal. S.B.Kasture, Sanjivani
`College of Pharmaceutical Education & Research,
`Kopargaon for providing us the required facilities to
`carry out the research work.
`
`References
`1. http://www.wikipedia.org/wiki/Lisinopril.
`29April2014
`2. Bhaskara Raju V. & Lakshmana Rao A. ,
`Novel Validated RP-HPLC Method For The
`Simultaneous Estimation Of Lisinopril &
`Amlodipine In Bulk & Tablet Dosage Form,
`IJPCBS 2011, 1(1), 32-37.
`3. Alaa El-Gindy, Ahmed A., Laila A., Marwan
`M.S., Spectrophotometric, Specroflurimetric &
`LC determination of Lisinopril, Journal of
`Pharmaceutical & Biomedical Analysis, 25
`(2001) 913-922.
`4. United States Pharmacopoeia 34-NF29(2011)
`Volume (3). Lisinopril dihydrate. USP-National
`Formulary Convension, Rockville, Maryland,
`3320.
`5. British Pharmacopoeia
`(2010) Volume
`Lisinopril Dihydrate. 1279-1280.
`6. Silverstein R.
` X (2005),
` M, Webster F.
`Spectrometric
` Identification
` of
` Organic
`Compounds, Published by, John Wiley & Sons
`Publications, sixth edition, 81-109.
`7. ICH, “Validation of Analytical Procedures;
`Text
` &
` Methodology,”
`
`International
`Conference
` on
` Harmonization
` Guidance
`Documents, Q2 (R1), 2005.
`8. International Conference on Harmonization
`(ICH) (1996) Stability Testing: Photostability
`Testing of New Drug Substance & Drug
`Products, Q1B.
`9. ICH, Stability Testing, Q1A (R2), Stability
`testing of New Drug Substances & products.
`Feb 2003; 1-20.
`10. Ramadevi
` et al.,
` Forced
` Bhimavarapu
`Degradation Study of Paracetamol In Tablet
`Formulation Using RP-HLPC, Bulletin of
`Pharmaceutical Research 2011;1(3):13-17.
`11. Ahuja S. (2006). Impurities Evaluation of
`Pharmaceuticals. New York:Marcel Dekker; 2-
`13
`12. Analytical Profiles of Drug Substances &
`Excipients, Volume 21, Klaus Florey, by
`Academic Press, INC. 233-269.
`
`
`II.
`
`Fig no.16 GC-MS spectra photolytic degraded
`product
`
`
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`Fig no.17 Fragmentation pattern for photolytic
`degradation product
`
`
`
`
`
`4. Conclusion
`The forced degradation of Lisinopril was
`carried out as per ICH guidelines. The structures of
`degraded products were established by UV, IR &
`HPLC. A stability indicating validated UV & HPLC
`methods were developed
`for quantification of
`degraded products of Lisinopril. The mechanism of
`degradation was proved by GC-MS studies.
`
`
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