The document describes the development and validation of UV spectrophotometric methods for analyzing risperidone and lacosamide. It discusses selecting analytical wavelengths, developing standard curves, and validating the methods by determining accuracy, precision, specificity, linearity, range and other parameters as required by ICH guidelines. Validation results for the risperidone and lacosamide methods such as recovery percentages between 98.4-99.8%, precision of 0.67-0.50%, linear ranges of 2-6 μg/ml and 12-40 μg/ml respectively are also presented. The developed and validated methods provide accurate and precise quantification of active pharmaceutical ingredients and finished dosage forms using UV spectrophotometry.
Analytical method development,validation by uv spectroscopy
1. METHOD DEVELOPMENT, OPTIMIZATION AND
VALIDATION BY USING INSTRUMENT (UV-VISIBLE
SPECTRO PHOTOMETer)
PRESENTED BY:
T. LAKSHMI BHAVANI
(2015MPH40023)
Under the guidance of:
Dr. A. SRIDEVI
SPMVV - TIRUPATHI
Pharmaceutical Analysis-1st year
II-semester
1
M.Pharm .,Ph.D
August 6, 2016
2. CONTENTS:
METHOD DEVELOPMENT
OPTIMIZATION
VALIDATION OF INSTRUMENTATION (PHARMACEUTICAL
DOSAGE FORMS,API,EXCIPIENTS)
UV-VISIBLE SPECTROPHOTOMETER
CONCLUSION
REFERENCES
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3. METHOD DEVELOPMENT
DEFINITION:
It is defined as a process of formulating the
materials, conditions and protocol for measuring the analyte.
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4. STEPS INVOLVED IN UV SPECTROMETER
SELECTION OF SOLVENT SYSTEM
SELECTION OF ANALYTICAL WAVELENGTH.
STUDY OF BEERS LAMBERT’S LAW
TO PERFORM ANALYSIS OF STANDARD LABORATORY
MIXTURE & TABLET FORMULATIONS BY PROPOSED
METHOD.
TO VALIDATE THE DEVELOPED METHODS BY USING
DIFFERENT VALIDATED PARAMETERS.
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5. STUDY OF BEERS-LAMBERT’S LAW:
The assay of single component sample, which contains other
absorbing substances, is then calculated from the measured
absorbance by using one of three principle procedures.
They are, use of –
standard absorptive value,
calibration graph &
single or double point standardization.
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6. 1. IN STANDARD ABSORPTIVE VALUE METHOD
The use of standard A(1%,1cm) or E values are used in
order to determine its absorptivity.
2.IN CALIBRATION GRAPH METHOD
The absorbance’s of a number of standard solutions of
the reference substance at concentrations encompassing the
sample concentrations are measured and a calibration graph
is constructed.
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7. 3.The single point standardization:
Procedure involves the measurement of the absorbance of a
sample solution and of a standard solution of the reference
substance.
The concentration of the substances in the sample is
calculated from the proportional relationship that exists between
absorbance and concentration.
C test=(A test X C std)/A std
where,
C test and C std are the concentrations in the sample
and standard solutions respectively and A test and A std are the
absorbance’s of the sample and standard solutions respectively.
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8. SELECTION OF ANALYTICAL WAVELENGTH:
Proper wavelength selection of the methods depends upon the
nature of the sample & its solubility.
To develop a rugged & suitable spectro photometric method
(UV), the analytical condition where selected after testing different
parameters such as diluents, buffer & buffer concentration.
Standard solution in UV spectrometer between 200 - 400nm
on spectrum mode, using diluents as a blank.
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9. VALIDATION:
It is defined as establishing documented evidences
which provides a high degree assurance that a specific process
will consistently produce a product meeting its pre-determined
specification & quality characteristics.
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11. ACCURACY:
The closeness of agreement between the value which is
accepted as a conventional true value or an expected
reference value , and the value found.
PRECISION:
It expresses the closeness of agreement between a series
of measurements obtained from the multiple sampling of the
homogenous sampling under the prescribed conditions.
Precision may be considered at 3 levels: repeatability,
intermediate precision, reproducability.
1.Repeatability : it expresses the precision under the same
operating conditions over a short interval of time.
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12. 2.Intermediate precision: It expresses with in laboratory variations :
different days, different analyst, different equipment
3.Reproducability: It expresses the precision between laboratories.
DETECTION LIMIT: It is the lowest amount of analyte in a sample
which can be detected but not quantitated as an exact value.
QUANTITATION LIMIT: It is the lowest amount of analyte in a
sample which can be quantitatively determined which suitable
accuracy and precision.
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13. SPECIFICITY: It is the ability to acsses the analyte in the presence
of components which may be expected to be present.
It includes impurities, degradants, matrix etc.
This definition has following implications:
Identification: To ensure the identification of analyte
Purity test: To ensure an accurate content of impurities of an analyte
i.e. heavy metals, residual solvent content.
Assay: To provide an exact result this allows an accurate statement
on content or potency of analyte in the sample.
LINEARITY: Its ability(with in a given range) to obtain test results
which are directly proportional to the concentration(amount) of
analyte in the sample.
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14. RANGE: It is the interval between upper and lower concentration
of analyte in the sample.
ROBUSTNESS: It is a measure of its capacity to remain
unaffected by small variations in method parameters and provides
an indication of its reliability during normal usage.
RUGGEDNESS: Reproducibility under normal but variable
laboratory conditions.
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15. DEVELOPMENT OF ANALYTICAL METHOD FOR
RISPERIDONE(API) BY UV-SPECTROPHOTOMETRY:
PREPARATION OF STOCK SOLUTION:
100mg of pure drug was weighed & transferred to 100ml
volumetric flask, add 50ml of 0.1N HCl and make up the
volume with 0.1 N HCl.
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16. SAMPLE SOLUTION:
2mg of RIS was weighed and transferred to 100ml volumetric
flask.
Add 20ml of 0.1 N HCl and sonicate for 5 min.
Volume was made up with 0.1 N HCl the above solution was
mixed well and filtered through Whattmann’s filter paper.
Dilutions were made with 0.1N HCl to attain a concentration of
4μg /ml.
6 replicates of analysis were carried out with sample weighed
individually.
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18. PRECISION & ACCURACY:
Precision and accuracy were determined with standard quality
control sample prepared in triplicate at different concentration
levels.
Precision of the assay was determined by repeatability and
intermediate precision, which was studied by comparing the
assays on 3different days, results are documented as SD & %
RSD.
Accuracy is the percentage of analyte recovered by assay from
a known added amount.
Data from 9determinations over 3concentration levels covering
the specified range.
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19. Where s = the standard deviation of absorbance of the sample.
m = the slope of the related calibration graphs.
LOD:
Lowest concentration of
analyte that an analytical
process can reliably
differentiate from
background levels.
LOD = 3s/m
LOQ:
Lowest concentration
of the standard curve
that can be measured
with an acceptable
accuracy, precision and
variability.
LOQ = 10s/m
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20. LINEARITY:
5 point calibration curve
were generated.
evaluated by least square
regression method using un
weighed data.
STABILITY:
The stability of
risperidone in 0.1 N
HCl solution was
studied by UV method.
Sample solutions were
prepared in triplicate
and stored at 4 & 250 C
for 30,60,90 & 120
min.
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21. RECOVERY STUDY:
To study the accuracy, precision and reproducability of
the proposed method and dosage forms recovery experiments
were carried out using standard method.
These studies were performed by addition of known
amount of pure RIS to the pre analyzed tablet formulation &
the mixtures analyzed using proposed techniques.
After parallel analysis the recovery results were
calculated using the related calibration equations.
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23. DEVELOPMENT AND VALIDATION SPECTROSCOPY
METHOD FOR ESTIMATION OF LACOSAMIDE IN
PHARMACEUTICAL DOSAGE FORMS:
DETERMINATION OF MAXIMUM WAVELENGTH:
50 mg of lacosamide was weighed accurately and transferred into
a 100ml vol, flask & dissolved in 50ml of acetonitrile :
water(25:75) by sonication and made up to volume with the same
solvent mixture to give a standard concentration of 500μg /ml
transfer 5ml of the above solution into 100ml vol. flask dilute
and made up to volume with the solvent mixture to a standard
concentration of 25μg/ml.
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24. This solution was scanned against blank for the entire UV-
visible wavelength 200-800nm.
Based on the spectrum λ max of 230 nm was selected for
further analysis.
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25. STANDARD PREPARATION:
50 mg of lacosamide was weighed accurately
Dissolve in 50 ml of acetonitrile:water (25:75%v/v) by sonication
Make up the volume with solvent mixture to give a standard
concentration 500μg/ml
Transfer 2.5,3.5,5,6 &8ml ..of the standard solution into 100ml
vol.flask make upto volume with the solvent mixture to get a series
of standards 12.5,17.5,25,30 &40 μg/ml
Absorbance of the these standards are measured at λmax of 230nm.
Standard curve was drawn by plotting concentration vs absorbance.
d
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26. SAMPLE PREPARATION:
50mg of Lacosamide in to 100 ml vol. flask sonicate for 30min.
With shaking and made up to volume with acetonitrile:
water(25:75% v/v)
Filter the solution through 0.45 μm membrane transfer 5ml of the
above solution in to 100ml volume flask and dilute to volume with
the same solvent.
The absorbance of the resulting solution was measured at 230nm.
The actual conc. Of the drug in the sample was determined from
standard curve.
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27. METHOD DEVELOPMENT
To develop a suitable UV method for the determination of
lacosamide different compositions of water,methanol,acetonitrile
were used as diluent.
Finally good recoveries were found with diluent of
acetonitrile:water(25:75 % v/v)
On scanning the standard solution against diluent in entire UV-
visible region of 200-800nm good response was found at 230nm
for 25μg/ml.
There is no interference from dilute through the entire UV
visible range of 200-800nm.
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28. METHOD VALIDATION:
The developed UV method was validated as per
ICH guidelines.
ACCURACY:
The method was determined by three conc.levels by
recovery experiments .
The recovery studies were carried out in triplicate
preparation collected from 20 tablets of lacosamide and
analyzed as per proposed method.
The % recoveries : 98.4-99.8 with an overall %RSD of
0.5.
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29. Recovery studies for Lacosamide by proposed method
% level recovery range % RSD at each level over all % RSD
50 99.0-99.8 0.4
100 98.4-98.8 0.2 0.5
150 98.6-99.2 0.3
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30. PRECISION:
Evaluated by 3 independent assays of test Lacosamide
sample.
Intermediate evaluated by different analysts and
different days in same day.
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31. METHOD PRECISION (INTER AND INTRA) FOR
LACOSAMIDE:
REPLICATES METHOD PRECISION
INTER DAY INTRA DAY
1 99.3 99.80
2 98.4 101.1
3 99.1 99.9
4 99.9 100.3
5 100.3 100.8
6 99.7 100.3
AVERAGE 99.45 100.37
SD 0.67 0.50
% RSD 0.67 0.50
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32. SPECIFICITY:
To check for the interference of blank at the working
wavelength blank was scanned from 200-800nm.
As there is no blank interference observed at the working
wavelength of 230nm.
The UV spectroscopic method presented in this study is
specific for Lacosamide.
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33. LINEARITY AND RANGE:
The standard curve was obtained in the concentration range of
12-40μg/ml.
This method was evaluated by linear regression analysis.
Slope, intercept and correlation coefficient of standard curve
was <0.999.
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34. CONCLUSION:
A simple, economic, precise, accurate method for
estimation of Lacosamide and risperidone in bulk and
pharmaceutical formulation was developed.
This developed method was validated according to
ICH guidelines.
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35. REFERENCES
Analytical &bio analytical techniques-siladitya behera
2012
Asian journal of pharmaceutical and clinical research
vol-6,suppl-3,2013
Principles of instrumental analysis- skoogDA, holler FJ,
6 th addition
International journal of pharmaceutical science & research
vol.1(2),2010,122-126
The Merck Index, Merck Research Laboratories division
of Merck and company, 13th ed, NJ, USA, 2001, 1627
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