The document discusses analytical method development for pharmaceutical formulations, highlighting the need for innovative techniques due to the lack of standardized procedures for certain drugs. It details various analytical techniques, including titrimetric, spectrophotometric methods, and chromatography, while emphasizing the importance of accuracy, precision, and validation in these methods. Examples of specific drug estimations using different methods are provided, illustrating the practical applications of these techniques.

1. ANALYTICAL METHOD DEVELOPMENT
1
Mr. Sagar Kishor Savale
[Department of Pharmaceutics]
avengersagar16@gmail.com
2015-2016
Department of Pharmacy (Pharmaceutics) | Sagar savale
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2. Need
» Herbal Products
» New process and reactions
» New molecules
» Active ingredients (Macro analysis)
» Residues (Microanalysis)
» Impurity Profiling
» Component of Interest in different matrices
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3. Outcomes
Analytical method gives-
» The required data for a given analytical problem
» The required sensitivity
» The required accuracy
» The required range of analysis
» The required precision
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4. Basic criteria
 The drug or drug combination may not be official in any pharmacopoeias.
 A proper analytical procedure for the drug may not be available in the
literature due to patent regulations.
 Analytical methods may not be available for the drug in the form of a
formulation due to the interference caused by the formulation excipients.
 Analytical methods for the quantitation of the drug in biological fluids may
not be available.
 Analytical methods for a drug in combination with other drugs may not be
available.
 The existing analytical procedures may require expensive reagents and
solvents. It may also involve cumbersome extraction and separation
procedures and these may not be reliable.
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5. Analytical techniques
 Titrimetric & gravimetric
 Colorimetric & ultraviolet spectrophotometric
 Electrochemistry
 Fluor metric
 Flame photometric & atomic absorption spectrometric
 Chromatography
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6. Spectrophotometric Methods of Analysis For
Drugs In Combination
 The basis of all the spectrophotometric techniques for multicomponent
samples is the property that at all wavelengths:
- the absorbance of a solution is the sum of absorbance of the individual
components or
- the measured absorbance is the difference between the total absorbance of
the solution in the sample cell and that of the solution in the reference cell.
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7. Nature of analysis
 Single component analysis
- Use of standard absorptivity value
- Use of calibration curve
-Single or double point standardization
 Multicomponent analysis
-Simultaneous equation method
-Two wavelength method
-The absorption ratio method
-Geometric correction method
-Absorption factor method
-Orthogonal polynomial method
-Difference spectrophotometry
-Derivative spectrophotometry
-Area under curve method
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8. Simultaneous Equation Method
Condition-
If a sample contains two absorbing drugs (X &
Y) each of which absorbs at the lambda max of
each other.
Equation -
Cx = (A2 ay1 - A1 ay2) / (ax2 ay1 - axay2)
Cy = (A1 ax2 - A2 ax1) / (ax2 ay1 - ax1ay2)
Where,
ax1 and ax2 - absorptivities of X at λ1 and λ2
respectively
ay1 and ay2 - absorptivities of Y at λ1 and λ2
respectively
A1 and A2 - absorbance of the diluted sample
at λ1 and λ2 respectively.
Cx and Cy be the concentration of X and Y
in the diluted samples respectively
8
Fig.1The overlain spectra of substance X and
Y, showing the wavelength for the assay of X
and Y in admixture by the method of
simultaneous equation.
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9. Examples of reported Drugs
- Estimation of Gliclazide and Metformin hydrochloride in
combined dosage forms.
- Estimation of Losartan potassium and Hydrochlorothiazide in
tablets.
- Estimation of Salbutamol and Theophylline from tablets.
- Estimation of Amlodipine besylate and Enalepril maleate from
tablets.
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10. Example
Ultraviolet Spectrophotometric determination of Olmesartan medoxomil &
Hydrochlorothiazide in pharmaceutical formulation by simultaneous equation
method
10
Fig.2 Overlay of Olmesartan medoxomil & Hydrochlorothiazide
Fig.3 Overlay of mixed sample of Olmesartan medoxomil & Hydrochlorothiazide
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11. Q-Absorbance Method (Absorbance Ratio
Method)
• The ratio of absorbance's at any two
wavelengths is a constant value
independent of concentration or path
length
• For example, two different dilution
of the same substance give the same
absorbance ratio A1/A2.
• In the USP, this ratio is referred to as
Q value
• Absorbance's are measured at two
wavelengths :-
-λmax of one of the component (λ2)
-wavelength of equal absorptivity's
of the two components i.e. an is
absorptive point. 11
Fig. 4 Wavelength for the assay of
substances X and Y in admixture by the
method of absorbance ratio method4/28/2016 Sagar Savale

12. Example
Estimation of Valsartan and Hydrochlorothiazide in pharmaceutical dosage
form by absorption ratio method 7
12
Fig. 5Overlay spectra of Valsartan & Hydrochlorothiazide showing iso-absorptive point at 231.5nm
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13. Q-Absorbance Method (Absorbance Ratio
Method)
Examples -
• - Estimation of Rifampicin and Isoniazid in pharmaceutical dosage forms.
• - Estimation of Spironolactone and hydroflumethiazide.
• - Estimation of Nalidixic acid and Metronidazole from tablets.
• - Estimation of Noscapine, Chlorpheniramine Maleate and Ephedrine
hydrochloride from tablets.
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14. Derivative Spectroscopy
• Derivative spectrophotometry involves the conversions of a normal
spectrum to its first, second or higher derivative spectrum.
• Advantages -
1.Enhanced resolution
2.Bandwidth discrimination
• Methods of derivative spectroscopy -
1.Modification of the optical system
2.Electronic differentiation of the spectrophotometer analogue signal
3.Microcomputer differentiation
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15. Examples of reported drugs
-Estimation of Propranolol and Hydrochlorothiazide.
-Estimation of Phenylpropanolamine, Chlorpheniramine and
Bromhexine.
-Estimation of Naphazoline hydrochloride and Chlorpheniramine
maleate.
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16. Derivative Spectroscopy (continue)
Development and validation of spectrophotometric method for simultaneous
estimation of Samaritan and Naproxen sodium in tablet dosage form 8
16
Fig .6 First derivative overlain spectra of SUM and NAP
Solvent : Methanol; Zero crossing point at 298nm & 335nm for SUM & NAP resp.
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17. Difference Spectroscopy
17
Fig.7 Difference spectrum of BSZ (10 μg/mL solution of BSZ in 0.1 M HCl was taken
as blank and the same concentration of drug in 0.1 M NaOH as sample).
Abs.
wavelength
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18. Multicomponent mode of analysis
18
Fig .8 UV spectrum of Ceftriaxone sodium and
Sulbactam sodium
Fig. 9Assay report of ceftriaxone sodium and
sulbactam sodium by multi-component mode
of analysis
Solvent : 0.1 Sodium Hydroxide ; Wavelenght :251nm(Sulbactum) & 259nm(Ceftriaxone)4/28/2016 Sagar Savale

19. Solvent Extraction Method
• Separation of individual drugs based on their selective solubility
followed by spectrophotometric measurement.
• Separate the absorbing interfering from the analyte by solvent
extraction procedure.
• Examples :-
-Estimation of Probenecid and Ampicillin from tablets.
-Estimation of Probenecid and Cephalexine from tablets.
-Estimation of Caffeine from Aspirin and Caffeine tablets.
-Estimation of Paracetamol and Diclofenac sodium from tablets.
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20. Chemical Derivatisation
 Indirect spectrophotometric assays
 Conversion of the analyte by a chemical reagent to a derivative that has
different spectral properties
 Reasons for adopting chemical Derivatisation
1.Weak absorption of the analyte
2.Interference from irrelevant absorption
3.Improve selectivity of the assay
4.Cost
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21. Chemical Derivatisation
Methods
1.Diazotisation & coupling of primary aromatic amines
2.Condensation reactions
3.Reduction of tetrazolium salts
4.Acid – dye method
5.Oxidation methods
6.Metal legand complexation
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22. Example
Development and validation of spectrophotometric method for estimating
Amisulpride in pharmaceutical preparations11
Diazotisation & coupling of primary aromatic amines
 Diazotization of Amisulpride with sodium nitrite and hydrochloric acid,
followed by coupling with -
-N-(1-naphthyl) ethylenediamine hydrochloride (Method A),
-diphenylamine (Method B),
- β-naphthol in an alkaline medium (Method C),
- resorcinol in an alkaline medium (Method D) &
- chromotropic acid in an alkaline medium (Method E) to form a colored
chromogen.
 The absorption maxima, λmax, are at
-523 nm for Method A,
-382 and 490 nm for Method B,
-527 nm for Method C,
-521 nm for Method D and
-486 nm for Method E.
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23. Area under curve method
23
Fig.10 Overlay spectrum of RAM and HCT
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24. Analytical method validation
 Accuracy
 Precision
 Linearity
 Range
 Limit of Quantitation
 Limit of Detection
 Ruggedness
 Robostnes
 Specificity
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25. References
1. Rashmin. An Introduction To Analytical Method Development For
Pharmaceutical Formulations .Pharma info.net 2008 July 22;6(4):1-27
2. Sethi PD.Quantitative Analysis of Drugs In Pharmaceutical
Formulations.3rd ed. New Delhi, CBS Publishers & distributors;2005, p.7
3. Saraf S. Various UV spectrophotometric simultaneous estimation
methods.Pharma info.net 2006 April 04;4(2)
4. Daharval SJ.Methods of estimation of multi -component formulations.
Pharma info.net 2006 June 19;4(3)
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