INTRODUCTION:
Quality level of any analytical work
in a quality
control laboratory depends on the expertise of the analyst, most
appropriate analytical procedures and overall performance of analytical instruments. The main task of pharmaceutical analyst is therefore to provide
reliable analytical data rapidly
and accurately.
Analytical5,6 research and development is a requisite part of pharmaceutical industry whose
goals include
contributing to the development of new active substances and pharmaceutical dosage forms
by providing information based on analytical chemistry, by developing analytical methods
and specifications used
in quality control
of material for toxicological and clinical trials, and by subsequent transfer of these
methods and specifications.
The development of a way for analysis of a sample should take
into consideration that
the analytical information is characterized with quality
and reliableness. The quality13-17 and reliableness are obtained provided that
the analyst is versatile in selecting simplest ways
for the sample
and for instruments used for the development. Quality
is must in each product; however, it is imperative in medication because it involves life. Quality
control could
be a thought
that strives to provide an ideal
invention by sequence of trails
as well as eliminate errors at different phases of manufacture.
The quality of the drug being
analyzed to replicate the standards associated with
efficacy, safety
and effectiveness. In analytical method development the sample
could be a crucial consideration.
The Qualitative and Quantitative analysis may be done by numerous analytical ways.
Quantitative analysis constitutes the most important part
of analytical chemistry with varied
methods and instrumentation used in determining the concentration of constituents in samples. It is one of the basic criteria in pharmacy where ever
quality is to be critically maintained.
Analytical instruments play
a key role
in the production, analysis of medication, as well
as the protection of consumers and surroundings. It provides the lower
detection limits
needed to assure safety of foods, drugs, water
and air. The manufacture of materials, whose composition should be monitored by analytical instruments. Instrumental methods are based on the theory of relationship between the content and physicochemical properties of system being analyzed. Simply the instrument is a part of entire
analysis. It is necessary to use many instrumental techniques to get the data
needed to solve an analytical problem. Instrumental methodology could
also be employed by analytical chemists to avoid wasting of time, to get increased accuracy.
Prasugrel1,2, 5-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4, 5, 6, 7- tetra
hydrate [3, 2-c] pyridin-2-yl acetate
Hydrochloride, is an inhibitor of platelet activation and aggregation through the irreversible binding of its active metabolite to the P2Y12 class of ADP receptors on platelets. The drug
is particularly effective in the treatment of serious
heart and blood vessel problems. It is currently the drug
of choice for controlling acute coronary syndrome3,4, Heart
attacks, strokes
and blood clots
in stents.
So, an accurate analytical method
is required to estimate Prasugrel in pharmaceutical formulation.
MATERIALS AND METHODS:
In
the present study following reagents and materials were employed for development
and validation of new UV- Spectrophotometric7-12 method for estimation
of Prasugrel in tablet dosage forms. The solvents utilized in the entire work were
MerkPvt.Ltd and all the chemicals were analytical grade.
Chemicals:
The
Prasugrel reference standard (assigned purity 98%) was obtained from Apotex, Bangalore.
The commercial pharmaceutical formulations were obtained from local pharmacies.
Methanol was procured from SD fine chemicals, India. Distilled water was prepared
by Aquatron deionizing water system.
Instrument
Used:
UV
spectrophotometer (Systronics)
Solubility studies:
Solubility studies for Prasugrel were performed by using
various solvents. The solubility of the drug was analyzed in distilled water, ethanol,
methanol, carbon tetrachloride, benzene, and chloroform and acetone and hydrochloric
acid.
Selection of Solvent:
Prasugrel hydrochloride was known to be soluble in solvents
like carbon tetrachloride, benzene, methanol, chloroform and acetone. The absorption
pattern of resulting solution is measured against respective blank solution in UV
range (200-400nm) and λmax was found in each solvent, and it was
compared with the UV cutoff of that particular solvent to avoid any interaction
between the sample peak and solvent peak.
Fig.
1: UV Spectrum of Prasugrel Hydrochloride (10 Ppm) in Methanol
In
case of methanol the interactions between solvent peak and sample peak were found
to be minimum compared to other solvents and hence methanol was selected as a solvent
for estimating the various parameters of Prasugrel hydrochloride.
Method
development for assay of Prasugrel:
Method
development for assay of Prasugrel tablets was initiated based on general method
development guidelines and literature.
Preparation of standard stock solution:
Standard stock solution is prepared by dissolving 10mg of Prasugrel hydrochloride in methanol and the volume was made
up to 100ml
to obtain 100ppm
solution and dilutions were made
as and when
required.
LINEARITY:
Aliquots
of standard solutions of Prasugrel hydrochloride ranging from 0.5 – 3ml were transferred
in to a series of 10ml volumetric flasks. The volume of each flask was made up to
the 10ml mark with methanol and the absorbances were measured at 218nm. A graph
was drawn by plotting concentration on X axis and absorbance on Y axis and the graph
has shown a good linearity with a correlation coefficient of 0.997.
VALIDATION
OF SPECTROPHOTOMETRIC METHOD:
1.
PRECISION:
A. REPEATABILITY:
From
standard Stock solution of Prasugrel hydrochloride 2ml were transferred in to 10ml
volumetric flasks. The volume was made up to the 10ml mark with methanol and the
absorbance was measured at 218nm.
B.
INTERMEDIATE PRECISION:
ANALYST
TO ANALYST:
Four
samples of 20ppm concentration of Prasugrel hydrochloride were prepared by four
different analysts and absorbance was observed under same experimental conditions.
EQUIPMENT
TO EQUIPMENT:
Three
samples of 20ppm concentration Prasugrel hydrochloride were prepared and absorbances
were observed using different equipment.
DAY
TO DAY:
Three
samples of 20ppm concentration of Prasugrel hydrochloride were prepared and absorbances
were observed. Again, three fresh samples of the same concentration were prepared
on the following day and absorbance was measured.
C.
REPRODUCIBILITY:
From
standard Stock solution of Prasugrel hydrochloride 2ml solution was transferred
in to three 10ml volumetric flasks. The volume was made up to the 10ml mark with
methanol and the absorbance was measured at 218nm. The above three samples of 20ppm
concentration of Prasugrel hydrochloride were observed in different labs.
2.
ACCURACY (RECOVERY):
The
accuracy of the method was determined by analyzing three solutions containing Prasugrel
hydrochloride in 75%, 100% and 125% of the standard taken (20ppm).
For
preparing 75% concentration of standard take 1.50 mg, 1.51mg, 1.50mg of standard
Prasugrel is taken into three 10ml volumetric flasks and make up to the mark with
methanol. For preparing 100% concentration of standard take 2.00mg, 2.10mg, 2.02mg
of standard Prasugrel is taken into three 10ml volumetric flasks and make up to
the mark with methanol. For preparing 125% concentration of standard take 2.50 mg,
2.51 mg, 2.51 mg of standard Prasugrel is taken into three 10 ml volumetric flasks
and make up to the mark with methanol.
3.
RUGUDNESS:
From
standard Stock solution of Prasugrel hydrochloride 2 ml solution was transferred
in to three 10 ml volumetric flasks. The volume was made up to the 10 ml mark with
methanol. The absorbance of 20 ppm Prasugrel hydrochloride solution was measured
at different wavelengths. (218 +/- 5 nm).
4.
LINEARITY RANGE:
The
linearity range of the standard can be found from linearity curve and accuracy data.
The results were found to be linear in the concentration range of 5 – 30ppm.
Preparation
of sample solution:
20
tablets of Prasugrel hydrochloride (EFIPLAT 10) were taken, and all the tablets
were crushed to fine powder using a mortar and pestle. Powder equivalent to 10mg
of Prasugrel hydrochloride was weighed and transferred in to a 100ml volumetric
flask. The contents were dissolved in methanol and sonicated for about 30 min. This
solution was filtered through What man filter paper and the volume was made up to
the mark using methanol and dilutions were made as and when required to get the
desired concentrations.
LINEARITY:
By
using the test method, the absorbance of solutions at different concentrations prepared
from tablet were measured and checked for their linearity, by taking absorbance
on Y axis and concentration in ppm on X axis a graph was constructed and it was
found to have a good linearity with a correlation coefficient of 0.9967.
Table
1: linearity of Prasugrel in sample
|
S. No.
|
Concentration ppm
|
Absorbance
|
|
1.
|
5
|
0.135
|
|
2.
|
10
|
0.256
|
|
3.
|
15
|
0.412
|
|
4.
|
20
|
0.561
|
|
5.
|
25
|
0.658
|
|
6.
|
30
|
0.798
|
Fig.
No 2: Prasugrel Calibration curve in sample
RESULTS
AND DISCUSSION:
Prasugrel
was analyzed by using proposed UV spectrophotometric method in pharmaceutical formulation.
It is soluble in methanol and hence it was selected as a diluent for Prasugrel to
obtain UV spectrum in the range of 400-200 nm. After the evaluation of the spectrum,
Prasugrel showed maximum absorption at 218 nm.
1.
LINEARITY:
Table
No. 3: Linearity values of standard Prasugrel
|
S. No.
|
Concentration ppm
|
Absorbance
|
|
1
|
5
|
0.176
|
|
2
|
10
|
0.327
|
|
3
|
15
|
0.47
|
|
4
|
20
|
0.628
|
|
5
|
25
|
0.763
|
|
6
|
30
|
0.875
|
Fig.
No 3: Prasugrel calibration curve
2. REPEATABILITY:
Table
No.: 4 repeatability values of Prasugrel
|
S. No.
|
Concentration ppm
|
Absorbance
|
|
1.
|
20
|
0.630
|
|
2.
|
20
|
0.629
|
|
3.
|
20
|
0.630
|
|
4.
|
20
|
0.628
|
|
5.
|
20
|
0.629
|
|
6.
|
20
|
0.628
|
Mean
= 0.6285
Standard
deviation = 0.0010
% Relative
standard deviation = 0.159
5.
RUGGUDNESS:
Table
No. 10: Ruggedness values of Prasugrel
|
S. No.
|
Concentration (ppm)
|
Wavelength (nm)
|
Absorbance
|
|
1.
|
20
|
213
|
0.626
|
|
2.
|
20
|
214
|
0.627
|
|
3.
|
20
|
215
|
0.626
|
|
4.
|
20
|
216
|
0.627
|
|
5.
|
20
|
217
|
0.627
|
|
6.
|
20
|
218
|
0.628
|
|
7.
|
20
|
219
|
0.628
|
|
8.
|
20
|
220
|
0.627
|
|
9.
|
20
|
221
|
0.626
|
|
10.
|
20
|
222
|
0.625
|
|
11.
|
20
|
223
|
0.625
|
Mean
= 0.626
Standard
deviation = 0.0010
% Relative
standard deviation = 0.159
6.
LIMIT OF DETECTION:
LOD
= 3.3 σ / S
=
3.3 x 0.001/0.028
= 0.1178
ppm
7.
LIMIT OF QUANTIFICATION:
LOQ
= 10 σ / S
= 10
x 0.001/0.028
= 0.3571
ppm
CALCULATION
FOR PRASUGREL:
Assay= SPL abs × STD wt × Spl Dilln × 100 × Avg wt × % potency
STD abs STD dilln Spl wt LC
100
= 0.639 × 10 × 10 × 100 × 188.5 × 98
0.600 10 20.8 90 100
=98.4%
ANALYTICAL PERFORMANCE PARAMETERS OF PRASUGREL:
Table
No. 11: Analytical performance parameters of Prasugrel
|
S. No.
|
Parameter
|
Value
|
|
1.
|
Linearity range
|
5 - 30 ppm
|
|
2.
|
Slope (m)
|
0.028
|
|
3.
|
Intercept (c)
|
0.042
|
|
4.
|
Correlation coefficient
|
0.997
|
|
5.
|
Standard deviation
|
0.001
|
|
6.
|
LOD
|
0.1178 ppm
|
|
7.
|
LOQ
|
0.3571 ppm
|
|
8.
|
Accuracy
|
98-102%
|
|
9.
|
assay
|
98.4%
|
SUMMARY
AND CONCLUSION:
SUMMARY:
An
UV Spectrophotometric method was developed and validated as per ICH guidelines.
To
optimize the diluents, various solvents were tested, the use of methanol resulted
in no interference at λmax 218nm.
By
applying the proposed method the λmax of Prasugrel hydrochloride
was found to be 218nm. The linearity was established by comparing various concentrations
and absorbance’s at absorption maximum. The linearity was found in the concentration
range of 5 – 30ppm.
The
limit of detection and limit of quantization values were found to be 0.1178ppm and
0.3571ppm. This indicates the sensitivity of the method. The high percentage recovery
indicates the proposed method is highly accurate. No interference peaks were found
near the λmax indicating the recipients used in tablet formulation
did not interfere with the estimation of the drug by the proposed UV method.
CONCLUSION:
A simple, rapid and precise UV spectrophotometric method
has been developed for the determination of Prasugrel in bulk and dosage form. The
developed method was validated for linearity, accuracy, precision, ruggedness, LOD,
LOQ parameters. The result of all these parameters shows that the rapid, accurate,
linear and precise. This method can be successfully applied for routine estimation
of Prasugrel in bulk pharmaceutical dosage form.
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1.
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Baker WL, White CM. Role of Prasugrel,
A Novel P2Y12 Receptor Antagonist, in the Management of Acute Coronary Syndromes.
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DOI: 10.5958/2231-5675.2020.00037.X