analytical method development

05/02/2023 Sagar Savale 1

ANALYTICAL METHOD DEVELOPMENT

Mr. Sagar Kishor Savale[Department of Pharmaceutics]

[email protected]

Department of Pharmacy (Pharmaceutics) | Sagar savale

mailto:[email protected]


Need

» Herbal Products

» New process and reactions

» New molecules

» Active ingredients (Macro analysis)

» Residues (Microanalysis)

» Impurity Profiling

» Component of Interest in different matrices


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


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.


Analytical techniques Titrimetric & gravimetric

Colorimetric & ultraviolet spectrophotometric

Electrochemistry

Fluor metric

Flame photometric & atomic absorption spectrometric

Chromatography


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.


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


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

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.


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.


Example Ultraviolet Spectrophotometric determination of Olmesartan medoxomil & Hydrochlorothiazide in pharmaceutical formulation by simultaneous equation method

Fig.2 Overlay of Olmesartan medoxomil & Hydrochlorothiazide Fig.3 Overlay of mixed sample of Olmesartan medoxomil & Hydrochlorothiazide


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.

Fig. 4 Wavelength for the assay of substances X and Y in admixture by the method of absorbance ratio method


Example Estimation of Valsartan and Hydrochlorothiazide in pharmaceutical dosage form by absorption ratio method 7

Fig. 5Overlay spectra of Valsartan & Hydrochlorothiazide showing iso-absorptive point at 231.5nm


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.


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


Examples of reported drugs

-Estimation of Propranolol and Hydrochlorothiazide.

-Estimation of Phenylpropanolamine, Chlorpheniramine and Bromhexine.

-Estimation of Naphazoline hydrochloride and Chlorpheniramine maleate.


Derivative Spectroscopy (continue) Development and validation of spectrophotometric method for simultaneous estimation of Samaritan and Naproxen sodium in tablet dosage form 8

Fig .6 First derivative overlain spectra of SUM and NAP

Solvent : Methanol; Zero crossing point at 298nm & 335nm for SUM & NAP resp.


Difference Spectroscopy

Fig.7 Difference spectrum of BSZ (10 μg/mL solution of BSZ in 0.1 M HCl was takenas blank and the same concentration of drug in 0.1 M NaOH as sample).

Abs.

wavelength


Multicomponent mode of analysis

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)


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.


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


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


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.


Area under curve method

Fig.10 Overlay spectrum of RAM and HCT


Analytical method validation Accuracy Precision Linearity Range Limit of Quantitation Limit of Detection Ruggedness Robostnes Specificity


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)

analytical method development

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