| Research Article |
Open Access |
|
| Bioequivalence Study of Simvastatin |
| Selvadurai Muralidharan1, Janaki Sankarachari Krishnan Nagarajan1*, Sachin Singh1, and Anil Dubala1 |
| 1Department of Pharmaceutical Analysis, J.S.S.College of Pharmacy, Tamilnadu, India |
| *Corresponding author: |
Dr. Janaki Sankarachari Krishnan Nagarajan,
Center for Advanced Drug Research and Testing, J.S.S.College of pharmacy,
Tamilnadu, India,
Tel: +91-423-2443393,
Fax: +91-423-2442937,
E-mail: nagasaki2001@rediffmail.com |
|
| |
| Received October 05, 2009; Accepted December 24, 2009; Published
December 24, 2009 |
| |
| Citation: Muralidharan S, Nagarajan JSK, Singh S, Dubala A (2009)
Bioequivalence Study of Simvastatin. J Bioanal Biomed 1: 028-032.
doi:10.4172/1948-593X.1000006 |
| |
| Copyright: © 2009 Muralidharan S, et al. This is an open-access article distributed under the terms of the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author
and source are credited. |
| |
| Abstract |
| |
| A simple, rapid and selective method was developed for estimation
of simvastatin from human plasma. The method involves
simple protein precipitation techniques using etofylline as internal
standard. Chromatographic separation was carried out on a
reversed phase C18 column using mixture of methanol: 2mM
ammonium acetate and 500 μl of 0.5% formic acid (80:20, v/v)
at a flow rate of 1.0 ml/min with UV-VIS detection at 418.35
nm. The retention time of simvastatin and internal standard were
5.41 and 1.086 min, respectively. The method was validated and
found to be linear in the range of 1.0-10.0 ng/mL. An open, randomized,
two-treatment, two period, single dose crossover,
bioequivalence study in 12 fasting, healthy, male, volunteers was
conducted. After dosing, serial blood samples were collected
for the period of 24.0 h. Various pharmacokinetic parameters
including AUC0–t, AUC0–∞ , Cmax, Tmax, T½, and elimination rate
constant (Kel) were determined from plasma concentration of
both formulations. Log transformed values were compared by
analysis of variance (ANOVA) followed by classical 90% confidence
interval for Cmax, AUC0–t and AUC0–∞ and was found to
be within the range. These results indicated that the analytical
method was linear, precise and accurate. Test and reference formulation
were found to be bioequivalent. |
| |
| Keywords |
| |
| Pharmacokinetic studies; Validation; HPLC;
Statistical analysis; Simvastatin |
| |
| Introduction |
| |
Simvastatin is a hypolipidemic drug belonging to the class of
pharmaceuticals called statins. It is used to control hypercholesterolemia
(elevated cholesterol levels) and to prevent cardiovascular
disease (Wikepedia). Simvastatin is a powerful lipidlowering
drug that can decrease low density lipoprotein (LDL)
levels by up to 50%. It is used in doses of 5 mg up to 80 mg.
Higher doses (160 mg) have been found to be too toxic, while
giving only minimal benefit in terms of lipid lowering. There is
no real effect on HDL and triglyceride levels. From recent research
it has become apparent that simvastatin and other statins
inhibit the progression of atherosclerosis beyond their effects on
LDL. Many explanations have been proposed, for example its
inhibitory effect on macrophages in the atherosclerotic plaque
lesions. In one non-randomized study, simvastatin halved the
risk of developing dementia or Parkinson’s disease (Wolozin et
al., 2007) Limited analytical methods have been developed for
the determination of simvastatin in biological samples. Along
them high-performance liquid chromatography methods (Barrett
et al., 2006; Carlucci et al., 1991; Hazem et al., 2009; Novakova
et al., 2008; Hefnawy et al., 2009; Vuletic et al., 2005). Therefore,
the aim of the present investigation was to develop a new,
sensitive HPLC method for the estimation of simvastatin in human
plasma. The method was applied to a bioequivalence study
of simvastatin 40 mg tablets. The outcome of a study depends upon the reliability, reproducibility and sensitivity of the analytical
methodology employed. Therefore, the bioanalytical
method was validated in accordance with USFDA guidelines
prior to the initiation of the study. |
| |
| Experimental |
| |
| Materials |
| |
| Working standard of Simvastatin with 99.96% purity was obtained
from German Remedies Ltd., Mumbai, India. Etofylline
(purity 99.56%) working standard was obtained from Cadila
Health Care Ltd., Ahmedabad, India). Acetonitrile (HPLC grade),
obtained from Qualigens Fine Chemicals, Mumbai and potassium
dihydrogen ortho phosphate, ortho phosphoric acid, methanol,
and trichloroacetic acid (all analytical grade reagent) were
purchased from S.D. Fine Chem. Ltd., Mumbai. In house mill Q
water was used throughout the study. Fresh frozen human plasma
used in the method development was obtained from the Vijay
Hospital, Ooty, and was stored at -20 ± 2°C until required. |
| |
| Instrumentation and chromatography |
| |
| The HPLC system consisted of a LC-MS 2010 A (Shimadzu
Ltd., Japan), Auto injector port with 10μl loop (Rheodyne, USA)
and UV-VIS detector (Shimadzu Ltd., Japan). The wavelength
of the detector was set at 418.95 nm. Detector output was quantified
on Lab Solution chromatography software. Separation was
carried out on a Princeton SHER C18, 4.6mm × 100 mm, Japan,
using mixture of methanol: 2mM ammonium acetate and 500 μl
of 0.5% formic acid (80:20, v/v) as a mobile phase, at a flow
rate of 1 ml/min. Total analysis time was 15 min. All analysis
was performed at oven temperature 30°C. |
| |
| Preparation of calibration standard |
| |
| Stock solutions of simvastatin and olanzapine (I.S) (1 mg/ml)
were prepared in mixture of water and acetonitrile (1:1 V/V)
and stored at 8oC. The stock solution of simvastatin was further
diluted with the mixture of water and acetonitrile to give series
of standard solutions. Calibration standard of simvastatin
(10, 20, 40, 60, 80, 100 ng/ml) were prepared by spiking appropriate
amount of the standard solution in blank plasma. |
| |
| Quality control standards |
| |
| Lowest quality control standards (LQC), median quality control
standards (MQC) & highest quality control standards (HQC)
were prepared by spiking drug free plasma with simvastatin to
give solutions containing 2.0, 6.0, and 8.0, ng/ml, respectively.
They were stored at -20 ± 2°C till analysed. |
| |
| Sample preparation |
| |
| To 0.5ml plasma sample containing simvastatin (calibration
standard), 0.5ml of internal standard (10.0μg/ml) was added and
followed by 0.5ml of 10% trichloroacetic acid was vortexed followed
by centrifugation at 4000 rpm for 15 min at 4°C. The supernatant
solution was separated and injected. None of the drug
free plasma samples studied in this assay yield endogenous interference
at these retention times (Figure 1). |
| |
|
Figure1: Typical chromatogram of sample solution.
|
|
| |
| Validation (CDER 2001) |
| |
| The selectivity of the method was checked for interference
from plasma. The standard curve consisting of five points ranging
from 1.0 to 10.0 ng/ml was developed. Quality control
samples i.e. LQC (2.0 ng/ml), MQC (6.0 ng/ml) and HQC (10.0
ng/ml) were used to determine the intra and inter-day precision
and accuracy of the assay. Peak area ratios of simvastatin to internal
standard were fit to linear equation (y = 0.0012x-2E-06)
and drug concentration in control samples along with the same
day standard curve samples were calculated using this equation.
For all the curves the correlation coefficients (r2) were never
lower than 0.9982. |
| |
| Clinical design |
| |
| The study protocol was approved by The Institution Ethics
Committee. Twelve healthy adult, male, human Indian subjects
with mean age group 22.67 ± 2.27 years and average weight
61.67 ± 6.89 kg were included in the study. Subjects were excluded
from the study if one of more of following criteria were
present at time of medical screening: allergic to simvastatin, history or clinical data of renal or liver disease, positive test for
hepatitis B, HIV, history of alcohol, drug addiction or donated
blood within 72 days prior to study. Test and reference formulation
of simvastatin 40.0 mg tablet were administered with 240
ml of water. The study was conducted according to the principles
outlined in the declaration of Helsinki. The study was
conducted as 12×2 single dose, randomized, open, and complete
crossover design. Volunteers were fasted overnight before
and 3h after drug administration. |
| |
| Blood sample (5 mL) were collected at 0.00 h and 0.5, 1.0,
1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 6.0, 8.0, 12.0 and 24.0 of post
drug administration through an indwelling cannula into
heparinised glass vials. After drug administration standard breakfast
and lunch were provided at 3, 6, 9 and 12 h of post dose.
The blood samples were immediately centrifuged, plasma was
separated and stored at -20 ± 2°C until analysed. After a washout
period of 7 days, the study was repeated in the same manner to
complete the crossover design. The plasma samples obtained at
various time intervals were analysed by the HPLC method developed. |
| |
| Pharmacokinetic analysis |
| |
| The plasma concentration profile obtained was fed into PK
solution, computer software on Microsoft excel®, to determine
the pharmacokinetic parameters. The maximum simvastatin concentration
Cmax and the corresponding peak time Tmax were determined
by the inspection of the individual drug plasma concentration–
time profiles. The elimination rate constant Kel was obtained
from the least-square fitted terminal log-linear portion of
the plasma concentration–time profile. The elimination half-life
T½ was calculated as 0.693/Kel. The area under the curve to the
last measurable concentration (AUC0–t) was calculated by the
linear trapezoidal rule. The area under the curve extrapolated to
infinity (AUC0–∞) was calculated asAUC0–t +Ct/Kel where Ct is
the last measurable concentration (Figure 2). |
| |
|
Figure2: Mean concentration-Time Curve for 12 volunteers (Reference and Test products).
|
|
| |
| Statistical analysis (Bolton et al., 2004) |
| |
| For the purpose of bioequivalence analysisAUC0–t, AUC0–∞ and Cmax were considered as primary variables. Bioequivalence of
two formulations was assessed by means of an analysis of variance
(ANOVA) for crossover design and calculating 90% confidence
interval of the ratio of test/reference using log transformed
data. The formulation was considered bioequivalent when the
difference between two compared parameters was found statistically
insignificant (p > 0.05) and confidence interval for these
parameters fell within 80–125%. |
| |
| Results and Discussion |
| |
| Selectivity |
| |
| Selectivity of the method described was investigated by screening
six different batches of human blank plasma. Under the proposed
assay condition simvastatin and internal standard had a
retention time of 5.056 and 1.086 min, respectively, rest of the
peaks were due to the plasma components. Simvastatin and internal
standard were very well resolved under the proposed chromatographic
conditions. |
| |
| Accuracy |
|
| |
| The mean percent accuracy of the proposed method was found
to be 97.93%. |
| |
| Precision |
| |
| Intra day precision for simvastatin was 1.887 ± 0.0721, 5.809 ±
0.1402, 9.851 ± 0.1256 for the spiked concentration at 2.0, 6.0
and 10.0 ng/ml and the percent coefficient of variation (%CV)
was 3.82, 2.41 and 1.28, respectively. Inter day precision for
simvastatin was 1.8466 ± 0.1063, 5.69 40 ± 0.1863 and 9.5362
± 0.2723 for the spiked concentration at 2.0, 6.0 and 10.0 ng/
ml and the percent coefficient of variation (%CV) was 5.75, 3.27
and 2.86, respectively. (Table 1). |
| |
Table1: Precision studies.
|
|
| |
| Linearity |
| |
| The linearity of each calibration curve was determined by plotting
the peak area ratio of simvastatin to internal standard verses
nominal concentrat ion of simvastat in. For lineari ty
study seven different concentration of simvastatin were analysed (1.0,
2.0, 4.0, 6.0, 8.0, 10.0ng/ml). The peak area response was linear
over the concentration range studied. Each experiment at all concentration was repeated three times on three separate days to
obtain the calibration data. The coefficient of correlation ‘r2’
was found to be 0.9982. The limit of quantification and limit of
detection were 5.0 and 1.0 ng/ml, respectively. |
| |
| Recovery |
| |
| The mean extraction recoveries of simvastatin determined over
the concentration of 2.0, 6.0 and 10.0 ng/mL were 98.35%,
97.35% and 986.09%. For the internal standard (200 ng/mL),
the mean extraction recovery was 97.93% (Table 2). |
| |
Table2: Recovery Studies.
|
|
| |
| Stability study |
| |
| Short-term and long-term stock solution stability study was
evaluated, which proved no significant deviation from normal
value when stored at 4°C. The stability of simvastatin in plasma
was determined by measuring concentration change in quality
control samples over time. Stability was tested by subjecting the
quality controls to three freeze-thaw cycles and compared with
freshly prepared quality control samples. As shown in (Table 3),
the mean concentration of simvastatin in quality control samples
did not change significantly within the time period under the
indicated storage conditions. Long-term stability studies results
conclude that simvastatin is stable in plasma matrix at least for
30 days when stored at -20 ± 2°C. |
| |
Table3: Stability of drug in plasma during storage and plasma handling.
|
|
| |
| System suitability |
| |
| System suitability test was performed daily before the run of
analytical batch to check detector response to the analyte. This
method showed a good ruggedness, in fact little change in mobile
phase ratio or normal laboratory condition of humidity, light,
and air exposure temperature did not influence the retention time
of simvastatin and internal standard. Both the formulations were
well tolerated by all the volunteers in both the phases of study.
No clinical adverse events occurred during the study. All calculated
pharmacokinetic parameter values were in good agreement
with the previously reported values. For bioequivalence evaluation,
Cmax, AUC0-t, and AUC0–∞ were considered as primary parameters.
The mean and standard deviation of these parameters
of the two formulations were found to be very close, indicating
that the plasma profiles generated by test formulation is comparable
to those produce by reference formulation. Analysis of
variance (ANOVA), after log transformation of the data, showed
no statistically significant (p > 0.05) difference between the two
formulations. The mean peak plasma concentrations for 40.0 mg
simvastatin tablet were found to be 70.0128 and 81.3045 ng/ml
for the reference and test formulations respectively. UC0–24 was
found to be 56.8765 and 67.1555 ng h/ml for the reference and
test formulations respectively. AUC0–∞ was found to be 70.0128
and 81.3045 ng h/ml for the reference and test formulations respectively.
The elimination rate constant Kel for the reference
and test formulation was found to be 0.0899 and 0.0899, respectively. |
| |
| Conclusion |
| |
| An HPLC–UV based method has been developed for quantification
of simvastatin in human plasma. The sensitivity and simplicity of the method makes it suitable for pharmacokinetic studies.
The statistical comparison of AUC and Cmax clearly indicated
no significant difference in the two formulations of 40.0
mg of simvastatin tablets. Ninety percent confidence interval for
the mean (T/R) of AUC0–t, AUC0–∞ and Cmax indicates that the
reported values were entirely within the bioequivalence acceptance
range of 80–125% (using log transformed data). Based on
these results a concise decision on bioequivalence was taken.
Hence it was concluded that formulation ‘Test’ is bioequivalent
with formulation Reference. |
| |
|
| References |
| |
- Ali H, Nazzal S (2009) Development and validation of a reversed-phase
HPLC method for the simultaneous analysis of simvastatin and tocotrienols
in combined dosage forms. J Pharm Biomed Anal 49: 950-956. » CrossRef » PubMed » Google Scholar
- Barrett B, Huclova J, Borek-Dohalsky V, Nemec B, Jelinek I, et al. (2006)
Validated HPLC–MS/MS method for simultaneous determination of
simvastatin and simvastatin hydroxy acid in human plasma. J Pharm Biomed
Anal 41: 517-526. » CrossRef » PubMed » Google Scholar
- Bolton (2004) Experimental Design in Clinical trail in: Pharmaceutical Statistics.
- Carlucci G, Mazzeo P, Biordi L, Bologna M (1991) Simultaneous determination
of simvastatin and its hydroxy acid form in human plasma by highperformance
liquid chromatogtaphy with UV detection. J Pharm Biomed
Anal 10: 693-697.
- Guidance for Industry, Bioanalytical Method Validation. Rockville, Maryland:
U.S. Department of Health and Human Services. Food and Drug Administration,
Center for Drug Evaluation and Research CDER 2001.
- Hefnawy M, Al-Omar M, Julkhuf S (2009) Rapid and sensitive simultaneous
determination of ezetimibe and simvastatin from their combination
drug products by monolithic silica high-performance liquid
chromatographic column. J Pharm Biomed Anal 50: 527-534. » CrossRef » PubMed » Google Scholar
- Novakova L, Satinsky D, Solich P (2008) HPLC methods for the
determination of simvastatin and atorvastatin TrAC. Trends in Analytical
Chemistry 27: 352-367. » CrossRef » Google Scholar
- Practical and Clinical Applications, vol. 80, 3rd ed., Marcel Dekker Inc.,
New York, p384-443.
- Vuletic M, Cindric M, Koruznjak JD (2005) Identification of unknown
impurities in simvastatin substance and tablets by liquid chromatography/
tandem mass spectrometry. J Pharm Biomed Anal 37: 715-721. » CrossRef » PubMed » Google Scholar
- Wolozin B, Wang SW, Li NC, Lee A, Lee TA, et al. (2007) Simvastatin is
associated with a reduced incidence of dementia and Parkinson’s disease.
BMC Med 5: 20. » CrossRef » PubMed » Google Scholar
- www.Freewikipedia.com
- Yang AY, Sun L, Musson DG, Zhao JJ (2005) Application of a novel ultralow
elution volume 96-well solid-phase extraction method to the LC/MS/
MS determination of simvastatin and simvastatin acid in human plasma.
J Pharm Biomed Anal 38: 521-527. » CrossRef » PubMed » Google Scholar
|
| |
| |
|
|
|
This Article |
DOWNLOAD |
|
CONTRIBUTE |
|
SHARE |
|
EXPLORE |
|
 |
 |
| |
|
| |
| |
| |
|
Untitled Document
|
|
|
|
|
