1. INTRODUCTION:
Pravastatin and valsartan
use for cardio vascular disease. Pravastatin is a HMG-CoA reductase inhibitors. It
is newer class of compound are the most effectious
and best tolerated hypolipidemic drug. Valsartan is a angiotensin (AT1)
antagonist. Valsartan is an antihypertensive agent. Both
pharmaceutical compositions for the prevention or treatment of cardiac failure,
the prevention of ischemic coronary heart disease or the prevention of the
recurrence of ischemic coronary heart disease. Pravastatin
and valsartan novel combination used in treatment of
cholesterol induce hypertension patented by Sankyo company ltd, US 10/977, 645.
A. Pravastatin(3,4)
Figure 1 : Chemical Structure of Pravastatin
Pravastatin acts as a lipoprotein-lowering drug
through two pathways. In the major pathway, pravastatin
inhibits the function of hydro xymethylglutaryl-CoA (HMG-CoA) reductase.
As a reversible competitive inhibitor, pravastatin sterically hinders the action of HMG-CoA reductase
by occupying the active site of the enzyme. Taking place primarily in the
liver, this enzyme is responsible for the conversion of HMG-CoA to mevalonate in the rate-limiting step of the biosynthetic pathway
for cholesterol. Pravastatin also inhibits the
synthesis of very-low-density lipoproteins, which are the precursor to low-density
lipoproteins (LDL). These reductions increase the number of cellular LDL
receptors, thus LDL uptake increases, removing it from the bloodstream. Overall, the result is a reduction in
circulating cholesterol and LDL. Pravastatin is a
White to yellowish–white powder or crystalline powder, having molecular weight
446.5gm/mol.
Valsartan
is an ARB that selectively inhibits the binding of angiotensin II to AT1, which
is found in many tissues such as vascular smooth muscle and the adrenal glands.
This effectively inhibits the AT1-mediated vasoconstrictive
and aldosterone-secreting effects of angiotensin II and results in a decrease
in vascular resistance and blood pressure. Inhibition of aldosterone secretion
may inhibit sodium and water reabsorption in the
kidneys while decreasing potassium excretion. It is chemically
3-methyl-2-[pentanoyl-[[4-[2-(2h-tetrazol-5-yl)phenyl]phenyl]methyl]amino]-butanoic
acid. Valsartan is a White Crystalline Powder having
molecular weight435.52g/mol.
B. Valsartan(5.6)
Figure
2: Chemical structure of Valsartan
The review of
literature regarding quantitative analysis of Pravastatin
and Valsartan revealed that no Simultaneous Equation
method attempt was made to develop analytical methods for Pravastatin
and Valsartan. Some spectrometric methods and
chromatographic methods have been reported for the estimation of the individual
and combination of drugs.(7-55)
The focus of the present study was to develop and validate a rapid, stable,
specific, and economic Spectroscopic method for the estimation of Pravastatin and Valsartan in
Synthetic Mixture.
MATERIAL AND METHODS:
APPARATUS ANDINSTRUMENT:
·
A
double beam UV/ Visible spectrophotometer
(Shimadzu
model 2450, Japan) with spectral
width of 2nm, 1 cm quartz cells
was used to measure absorbance
of all the solutions.
·
Spectra were
automatically obtained
by UV-Probe system software.
·
An analytical
balance (SartoriusCD2250,
Gottingen, Germany) was
used for weighing the samples.
·
Sonicator (D120/2H,
TRANS-O-SONIC)
·
Class
‘B’ volumetric glassware were used (Borosillicte)
·
All instruments and glass wares
were calibrated.
REAGENTSAND
MATERIAL:
·
Pravastatin (Gift sample from
Ranbaxy Laboratories Ltd, Ahmedabad)
·
Valsartan (Gift sample from
Cipla Pharmaceuticals, Ankleswar)
·
Methanol
AR Grade (Finar), Distilled Water, NaOH AR Grade (Ranchem), HCl (Astron) was used for development purpose.
FIRST DERIVATIVE CONDITIONS:
·
Mode
: Spectrum
·
Scan speed
:Fast
·
Wave length
range: 200-400
nm
·
Derivative order: first
·
Scaling factor:
1
PREPARATIONOFSTANDARDSOLUTIONS:
Standard
solution of Pravastatin
(PRA):
Preparation
of stock solution of PRA:
Accurately
weighed quantity of PRA 10 mg was transferred to 100ml volumetric flask,
dissolved, and diluted up to mark with methanol to give a stock solution having
strength 100µg/ml.
Standard
solution of Valsartan (VAL):
Preparation
of stock
solution of VAL:
Accurately
weighed quantity of VAL 10 mg was transferred into 100 ml volumetric flask,
dissolved and diluted up to mark with methanol to give a stock solution having
strength 100µg/ml.
Preparation of Standard Mixture Solution (PRA + VAL):
1ml of
standard stock solution of PRA (100μg/ml) and 1ml of standard stock
solution of VAL (100μg/ml) were pipetted out
into two 10ml volumetric flasks and volume was adjusted to the mark with
methanol to get 10μg/ml of PRA and 10μg/ml of VAL.
Preparation of test solution:
The
preparation of synthetic mixture was as per patent:
·
Pravastatin:
10mg
·
Valsartan:
40mg
·
Microcrystalline
cellulose: 10 mg
·
Lactose
monohydrate: 10 mg
·
Povidone
K30: 10 mg
·
Croscamellose sodium: 10 mg
·
Magnesium
stearate: 10 mg
All the excipients were mixed in
100ml volumetric flask dissolved in 25 ml of methanol and sonicated
for 15min. make up the volume with methanol up to 100 ml. The solution was
filtered through Whatman filter
paper No.
42. Finally the solution had concentration 100μg/ml
for PRA and 400μg/ml
for VAL.
VALIDATION
OF PROPOSED METHOD:
Parameters to be considered for the validation of methods are:
1) LINEARITY ANDRANGE:
Procedure:
The linearity
response was
determined by
analyzing
5 independent levels
of calibration curve in the range of 2-10μg/ml and
8-40μg/ml for
PRA and VAL respectively
(n=6).
Calibration
curves
for PRA:
This series
consisted of five concentrations of standard PRA solution ranging from
2-10μg/ml. The solutions were prepared by
pipetting out
Standard PRA stock solution (0.2ml, 0.4ml, 0.6ml, 0.8ml,
1ml) was transfer red into
a series of 10ml volumetric
flask and volume was adjusted
up to mark with Methanol. A zero
order
derivative spectrum of the resulting solution was
recorded and
processed to first derivative
spectra,
measured
the absorbance at
248.20nm again stare agent
blank
solution (Methanol). Calibration curve
was
prepared by
plotting absorbance versus respective
concentration of PRA.
Calibration
curve for
VAL:
This series consisted
of five concentrations of standard VAL solution ranging from
8-40μg/ml.
The solutions were prepared
by pipetting out Standard VAL stock solution (0.8ml, 1.6ml, 2.4ml, 3.2ml, 4ml)
was transfer red
into a series of 10ml volumetric flask
and volume was adjusted upto mark with Methanol. A zero
order
derivative spectrum of the resulting solution was
recorded and
processed to first derivative
spectra, measured the absorbance at
262.40 nm against a reagent
blank
solution (Methanol). Calibration curve
was
prepared by
plotting absorbance versus respective
concentration of VAL.
2) PRECISION:
I. Intraday precision Procedure:
·
The precision
of the developed method was assessed
by analyzing samples of the same batch in nine
determinations with three Standard solutions containing
concentrations
2,4,6μg/ml for PRA and 8,16,24μg/ml for VAL and three replicate (n=3) each on same day.
·
First-derivative
absorbance (D1) was
measured at
248.20nm for PRA and 262.40 nm for VAL.
·
The %
RSD value of the results corresponding to the absorbance was
expressed for
intra-day precision.
II. Interday
Precision Procedure:
·
The precision
of the developed method was assessed
by analyzing samples of the same batch in nine
determinations with three Standard solutions containing
concentrations
2,4,6μg/ml for PRA and 8,16,24μg/ml for VAL and three replicate (n=3) each on different day.
·
First-derivative
absorbance (D1) was
measured at
248.20nm for PRA and 262.40nm for VAL.
·
The%
RSD value of the results corresponding to the absorbance was
expressed for
inter-day precision.
3) ACCURACY:
It was
determined by
calculating the recovery of
PRA and VAL by standard
addition method. Accuracy
was done by adding both API standard solution and test solution. Total
concentration was as per table 1
Procedure
Table 1:Solutions for Accuracy
Study
|
Concentration
of Formulation
(µg/ml) |
Concentration of API in spiking solution (µg/ml) |
Total concentration
of (μg/ml) |
|||
|
PRA |
VAL |
PRA |
VAL |
PRA |
VAL |
|
2 |
8 |
1.6 |
6.4 |
3.6 |
14.4 |
|
2 |
8 |
2.0 |
8 |
4 |
16 |
|
2 |
8 |
2.4 |
9.6 |
4.4 |
17.6 |
Each solution
was taken and diluted with Methanol up to 10ml volumetric flask and scanned between
200nm to 400nm against Methanol as blank. The amount
of PRA and VAL was calculated
at each
level
and % recoveries were computed.
4) LOD (LIMIT OF DETECTION) and LOQ (LIMIT
OF QUANTIFICATION):
The Limit of detection and Limit
of Quantification of the developed method was assessed by
analyzing
ten replicates of standard solutions containing concentrations
2μg/ml for PRA and 8μg/ml for VAL.
The LOD and LOQ maybe calculated as
Where, SD
=ten replicates of absorbance
Slope =the mean slope of the 6 calibration
curves
5) ROBUSTNESS ANDRUGGEDNESS:
Robustness and Ruggedness
of the method was determined
by subjecting the
method to slight change in the method
condition, individually,
the:
Ø Change in Stock Solution Preparation,
·
Stock-1
(10mg PRA in 100ml Methanol -100μg/ml
and 10mg VAL in 100ml Methanol - 100μg/ml)
·
Stock-2
(10mg PRA in 50ml Methanol -200μg/ml
and 10mg VAL in 50ml Methanol - 200μg/ml)
Ø Change in instrument
(UV-Vis
Spectrophotometer
model 1800 and 2450),
Three replicates
were made for the concentration (2, 4, 6 μg/ml of PRA and
8, 16, 24μg/ml
of VAL) with different stock solution preparation and the recording
of absorbances were done on both the UV-Vis spectrophotometer. %RSD
was calculated.
ANALYSIS OF PRA AND VALIN SYNTHETIC MIXTURE:
Composition
of synthetic mixture:
The
preparation of synthetic mixture was as per patent:
·
Pravastatin
: 10mg
·
Valsartan:40mg
·
Microcrystalline
cellulose: 10 mg
·
Lactose
monohydrate: 10 mg
·
Povidone
K30: 10 mg
·
Croscamellose sodium: 10 mg
·
Magnesium
stearate: 10 mg
All the excipients were mixed in
100ml volumetric flask dissolved in 25 ml of methanol and sonicated
for 15min. make up the volume with methanol up to 100 ml. The solution was
filtered through Whatman filter
paper No.
42.Finally the solution had concentration 100μg/ml
for PRA and 400μg/ml
for VAL. From that pipette out 1 ml in 10 ml volumetric flask and volume
was made up to mark with methanol to obtain final solution
containing
10µg/ml of PRA and
40µg/ml of VAL. A zero order
derivative spectrum of the resulting solution was
recorded
and processed to first derivative
spectra. A first order
derivative spectrum of the sample solution
was recorded
and the absorbance
at 248.20 nm and 262.40 nm were noted for estimation of PRA and VAL, respectively.
The concentrations of PRA and VAL in formulation were determined
using the corresponding calibration graph.
RESULT AND DISCUSSION:
Selection of
Wavelength and
Method Development
for Determination
of Pravastatin and
Valsartan:
The standard solution
of PRA and VAL were scanned
separately
between 200-400nm,
and zero-order spectra were not showed over lapping peaks.
Figure
3: Overlain zero order spectra of PRA and VAL, respectively
Thus
obtained spectra were then processed to obtain first-derivative spectra. First
order
derivative spectrum for PRA showed zero
crossing point: 262.40nm. The wave length
selected for estimation of PRA was 248.20 nm because it showed r2>0. 999 at this
wave
length in mixture.
First
order derivative spectrum for
VAL showed zero crossing point:
248.20nm. The wave length selected for estimation of VAL was 262.40 nm because it showed r2>0.9998 at
this wave length in mixture (Figure 3)
Figure 4: Overlain first order
spectra
of PRA and VAL
Figure 5: Overlain first order
spectra
of PRA and VAL in 3:1 ratios,
respectively
with the combination solution
(3:1)
The overlain
first order spectra (fig.3) of PRA and VAL reveal that PRA showed zero crossing at
262.40 nm, while VAL showed
zero
crossing at 248.20nm. At zero crossing
point (ZCP) of PRA (262.40 nm), VAL showed an absorbance, whereas at
ZCP of VAL (248.20 nm), PRA absorbance.
Hence
248.20 nm and 262.40 nm were
selected as analytical
wave
lengths for determination of Pravastatin and Valsartan, respectively.
Figure 6 : Overlain linear first
order spectra of PRA
(Green) and VAL (Red) in 1:4 ratios
Figure7 : Calibration
curve for
PRA at 248.20 nm and VAL at 262.40 nm
VALIDATION
PARAMETERS(59-57)
1. Linearity and Range:
The First-derivative
spectra (fig.3) showed linear absorbance at 248.20nm (ZCP of VAL) for PRA (2-10µg/ml)
and 262.40 nm (ZCP of
PRA) for VAL(8-40µg/ml) with correlation
coefficient (r2) of 0.999 and 0.9998 for PRA and VAL, respectively.
This method obeyed beer’s law in the concentration range
2-10 µg/ml and
8-40µg/ml for PRA and VAL, respectively. (Table2) Correlation coefficient (r2) from calibration
curve
of PRA and VAL was found
to be 0.999 and 0.9998, respectively. The regression line equation for PRA and VAL are as following,
y =
-0.0042x +0.0004 for PRA _____________ (1)
y =
-0.0009x +0.0008 for VAL ______________ (2)
From the combination
solution
of PRA and VAL the dilution
were
made in ratio of 1:4 and absorbance
were recorded
(Table 2) and correlation coefficient
(r2) of 0.999 and
0.9998 (figure 7) for PRA
and VAL, respectively.
Table
2: Calibration data for
PRA and VAL at 280.47 nm and
351.0nm, respectively. *(n=6)
|
Sr. No |
Concentration (μg/ml) |
Absorbance* (248.20 nm)±SD PRA |
Absorbance* (262.40 nm) ±SD VAL |
|
|
|
PRA |
VAL |
||
|
1 |
2 |
8 |
-0.0091±0.0011 |
-0.006±0.0014 |
|
2 |
4 |
16 |
-0.0171±0.0020 |
-0.0133±0.0018 |
|
3 |
6 |
24 |
-0.0253±0.0019 |
-0.020±0.0010 |
|
4 |
8 |
32 |
-0.0348±0.0019 |
-0.0271±0.0021 |
|
5 |
10 |
40 |
-0.0425±0.0052 |
-0.0338±0.0024 |
2. Precision:
I. Intraday precision:
The data for
intraday precision
for combined standard solution
of PRA and VAL is presented in Table 3. The % R.S.D was
found to be 0.63 - 0.84% for PRA and 0.49 – 0.81 % for VAL. These
% RSD
value was found to be less than ±2.0
indicated
that the method is precise.
Table 3: Intraday precision data for estimation of PRA and VAL* (n=3)
|
Conc. (μg/ml) |
Abs.* (PRA) Avg. ± SD (248.20 nm) |
% RSD |
Abs.* (VAL) Avg.± SD (262.40 nm) |
% RSD |
|
|
PRA |
VAL |
|||||
|
2 |
8 |
-0.0090±0.00005 |
0.63 |
-0.0070±0.00005 |
0.81 |
|
4 |
16 |
-0.0180±0.0001 |
0.84 |
-0.0141±0.0001 |
0.70 |
|
6 |
24 |
-0.0252±0.0002 |
0.79 |
-0.0201±0.0001 |
0.49 |
II. Interday
precision:
The data for
inter
day precision for combined
standard solution
of PRA and VAL is presented in Table 4. The% R.S.D was
found to be 0.64-0.99% for PRA and0.52-0.82 % for VAL. These % RSD value
was found to be less than ±2.0 indicated that the method is precise.
3. Accuracy:
Accuracy
of the method was determined
by recovery
study from synthetic mixture at
three
levels
(80%, 100%, and 120%) of standard
addition. The % recovery values
are tabulated in Table 5 and 6. Percentage
recovery for PRA and
VAL by this method was found in
the range of 99.54% -101.66% and
99.27%-99.75%, respectively,
The value of % RSD within
the limit indicated that the method is accurate
and percentage
recovery shows
that there is no interference from
the
excipients.
4. Limit
of detection
and
quantitation:
The LOD for PRA and
VAL was conformed to be 0.054 µg/ml
and
0.024µg/ml, respectively.
The LOQ for PRA and VAL was
conformed to be 0.166µg/ml
and 0.074µg/ml, respectively. The obtained LOD and LOQ results
are presented in Table 7.