Drug analysis (2)

Question 1

Some causes for deviation from the Beer-Lambert law

Sample

Answer 1

• Contamination
• Precipitation
• Degradation (photolysis)
• Fluorescence
• Tautomerisation
• pH effects
• Temperature

Question 2

Some causes for deviation from the Beer-Lambert law

Other

Answer 2

• Stray light
• Non-monochromatic light source
• Mismatched cells
• Sensitivity A-0.002
• Solvent absorption
• NB- Must establish the validity of the Beer-Lambert law for each drug under the measurement conditions to be used over an appropriate concentration range => calibration curves

Question 3

Calibration curves

Answer 3

• Use at least 5 standard solutions spanning the working concentration range
• Measure in duplicate in a matched pair cells against the solvent as reference
• Ideal absorbance range optimum for a modern spectrophotometer: 0.1-1.0
• Exceptionally this can be pushed up to an absorbance 1.5

Question 4

Instrumentation- single-beam spectrophotometers

Answer 4

• Intense source of UV light (Deuterium or hydrogen lamp)
• Prism of diffraction grating monochromator
• Capable of high precision (0.1-1.0 abs.unit)
• Used for absorbance determination at a fixed wavelength, not to obtain a spectrum

Question 5

Instrumentation: double-beam spectrophotometers

Answer 5

• Similar to single beam instrument but
  
  • Radiation split into 2 beams by rotating merror
  • One beam passes through the sample
  • The other beam passes through the reference cell
  • The 2 beams are compared to give the absorbance suitable for fixed wavelength readings and whole spectra

Question 6

Spectral bandwidth

Answer 6

• In some assays the minimum desirable resolution must be specified since changes in the spectral bandwidth (or monochromator slit width) can effect the absorbance of sharp peaks
• The BP (1980)- the spectral bandwidth used should be such that further reduction does not give an increase in absorbance
• Important for drugs having aromatic or strongly conjugated systems e.g. diphenhydramine and phenoxymethylpenicillin

Question 7

Stray light

Answer 7

• Increase with instrument age
• Needs to be checked
• BP (1980)- absorbance at 200nm of a 1.2% w/v KCl aq must be >2 absorbance units relative to water as a reference

Question 8

Quantitative applications

Answer 8

Pharmacological applications

• Single drugs
• Mixtures of drugs
• Colourimetric methods
• Tablet dissolution
• Limit tests for impurities
• Assays for bulk drugs or extracts

Other applications- physicochemical measurements

• pKa
• Velocity constants in enzymatic reactions

Question 9

Single component systems

Answer 9

• Single component systems
  
  • Establish linear range for compliance with the Beer-Lambert law using the centre of a broad maximum (calibration curve)
  • Adjust the drug concentration within the optimum instrument range
  • Measure the sample under identical conditions to the reference
• Problem- non-specific absorbance =>
  
  • Difference spectrophotometry
  • Second derivative spectrophotometry
  • Chemical or physical transformation of the drug to shift the lambda max to a longer wavelength

Question 10

Multicomponent systems

Answer 10

• The absorption spectra often overlap
• If the components obey the Beer-Lambert law and the law of additivity of absorbance applies then (see equation)

Question 11

Multicomponent systems

Answer 11

• Simultaneous equations (one per component)
• Need
  
  • Accurate absorptivity values
  • Non-overlapping lambda max regions for the components
  • The errors are very great for similar components
  • Make derivatives for coulourimtric analysis

Question 12

Derivative for colourimetric analysis

Answer 12

*

Question 13

Derivative spectroscopy

Answer 13

• The absorbance (A) of a sample is differentiated with respect to wavelength (lambda) by computer
  
  • Zero order- A=f(lambda)
  • 1^st derivative- dA/d(lambda)= f(lambda)
  • 2^nd derivative- dA²/d(lambda)²= f(lambda)
• Subtle changes of gradient in the normal spectrum (zero order) are observed as distinctive bipolar features
• Broad bands are suppressed relatice to sharp bands
  
  • Increases with increasing order of differentiation
  • This give selective rejection of broad additive spectral interferences such as rayleighs scattering

Question 14

Derivative spectroscopy

Answer 14

• First derivative
  
  • Represents the gradient at all points
  • Can locate ‘hidden’ peaks (dA/d(lambda)= 0 at peak maxima
• Even-order derivatives
  
  • The bipolar function of the alternating sign at the centroid (2nd- 4th+)
  • Centroid coincides with the original peak maximum
  • Centroid peak with decreases with increasing order of differentiation (useful for resolution of overlapping peaks)
  • Satellite peaks become increasingly complex

Question 15

Example 1

Answer 15

Question 16

Example 2

Answer 16

Question 17

Fluorescence spectrophotometry

Answer 17

• Very sensitive- better than absorption spectrophotometry (I_o/I difficult at low concentrations)
• Measured against a dark background
• Selective- fluorescent drugs and their metabolites may be analysed more readily than by conventional spectrophotometry
• Format
  
  • Scan Lambda_emmisions at fixed Lambda_excitation
  • Scan Lambda_excitation at fixed Lambda_emmision
  • Scan Lambda_excitation and Lambda_emmision synchoronously

Question 18

Fluorescence spectrophotometry

Signal intensity

Answer 18

• Generally more sensitive to the environment than absorbance measurements
• The signal intensity may be affected by
  
  • pH (ionisable groups)
  • Temperature (T, increased collisional quenching- use a thermostat)
  • Quenching (formation of complexes between the sample and another species
  • Interfering substances- often limiting in the analysis of biological samples; can be reduced by pre-treatment of the sample, use pure solvents, clean glassware
  • Solvents
  • Interference from Rayleigh and Raman scattering

Question 19

Quantitative applications of fluorescence spectrophotometry

In dilute solution

Answer 19

• The total absorbance of the system (Epliso b c) must not exceed 0.005 absorbance units
• At high drug concentrations, ground-state molecules may absorb the fluorescence emitted by excited molecules
  
  • => negative deviations from linearity