Quality Control The pharmacists are always in need for a method guaranteeing their drug quality and effectivity. Quality control of drugs requires marked abilities for analytical thinking as well as skills in experimental work.
Adequate facilities, approved procedures should be available for inspecting and testing starting materials, packaging materials, intermediates, bulk, and finished Products.
There are some problems in drug analysis resulted from: • 1-Interference from coexisting drugs and excipients. • 2-Interference from degradation products. • 3-Interference from endogenous compounds for the analysis of drugs in body fluids
The general method to be adapted for the analysis of mixtures is to separate them into their components and identify each component by 1- Physical methods 2- Physico-chemical methods 3- Chemical methods
Advanced Instrumental Analysis The course deals with the principles and applications of modern analytical instruments. Emphasis is placed upon the theoretical basis of each type of instrument, its optimal area of application, its sensitivity, its precision, and its limitations.
Spectrophotometry Photometric techniques are among the most important instrumental techniques available to the pharmaceutical analyst. The basis of all these instrumental techniques is that they measure the interaction of electromagnetic radiation with matter in specific energy levels.
Atomic spectra:- When certain compounds are heated, light of characteristic colors in emitted. For example sodium salts emit yellow light and potassium salts emit lilac light. This is due to the emission of light at wave lengths characteristic of the metallic elements in the sample. This is the simplest quantitative analytical procedures.
ΔE = Eu– El = hc / λ h = planck's constant = 4.132 × 10-15 eVs C = speed of light = 3 × 108 ms-1 in vacuo
Molecular spectra:- Molecular spectra are characterized by the absorption or emission of light over a much wider range of wave length called spectral bands than atomic spectra. This is due to the very large number of transitions which molecules can undergo in comparison to relatively few electronic transitions of atoms.
The total energy of a molecule is the sum of electronic, vibrational and rotational energies. The relative energy required to induce electronic vibrational and rotational transitions between quantized energy levels are approximately 10,000: 100:1 respectively
After absorption of light the excited molecules are short-lived and deactivation occurs due to:- •Internal collisions (internal conversion). •Cleavage of chemical-bonds, initiating photochemical reactions. •Re-emission as light (luminescence)
I = Io - IT %T = 100 IT/ Io Lambert (1760) found that there is a relation between the thickness of the media and the incident light ln Io / IT = K' b log Io / IT = K' b/ 2.303 A= log 1/T = log Io / IT =2 – log (%T)
Beer (in 1852) showed that there is a similar relationship between absorbance and the concentration. ln Io / IT = K''c K'' = proportionality constant. c = is the concentration. log Io/ IT = K''c / 2.303 log Io / IT = K' b/ 2.303 A = log Io / IT = a b c
A = Є b c Є = A1% x molecular weight/10 A = A1% 1cm b c Calculate the concentration of methyl testosterone in an ethanol solution of which the absorbance in a 1 cm cell at its λmax was found to be 0.890. The A1%1cm in the B.p. monograph of methyl testosterone is given as 540 at 241 nm.
The absorbance values at 250 nm of five standard solutions, a blank solution, and a sample solution of a drug are given in the table below; calculate the concentration of the sample. Concentration (µg ml-1) (x) A 250nm (y) 0 0.002 10 0.168 20 0.329 30 0.508 40 0.660 50 0.846 Sample 0.611
The λmax of the ephedrine hydrochloride and chlorocresol are 257 nm and 279 nm and the A1%1cm values in 0.1M hydrochloric acid solution are:- Ephedrine hydrochloride at 257 nm = 9.0 Ephedrine hydrochloride at 279 nm = 0 Chlorocresol at 257 nm = 20.0 Chlorocresol at 279 nm = 105.0 Calculate the concentrations of ephedrine hydrochloride and chlorocresol in a batch of ephedrine hydrochloride injection, diluted 1 to 25 with water, giving the following absorbance values in 1 cm cells. A279 = 0.424 A257 = 0.972
The concentration of a component in a sample which contains other absorbing substances may be determined by a simple spectrophotometric measurement of absorbance, provided that the other components have a sufficiently small absorbance at the wavelength of the measurement.
An example of this approach is the assay of paracetamol in pediatric paracetamol elixir. At the large over all dilution (approximately 3250 time) of the sample, the absorbance of interferents is negligible. If the identity, concentration and absorptivity of the absorbing interferents are known, it is possible to calculate their contribution to the total absorbance of a mixture.
Chemical derivatization :- The majority of indirect spectrophotometric procedures involve the conversion of the analyte to a derivative that has a longer λmax and or higher absorptivity. *If the analyte absorbs weakly in the ultraviolet region *The interference from irrelevant absorbance may be avoided by converting the analyte to a derivative
Diazotization and coupling of primary aromatic amines:- Ar – NH2 + HNO2 + H Ar – N+ N + 2H2O Ar - N+ N + Ar' – M Ar – N = N – Ar' + M+
Body fluids The determination of drugs and metabolits in body fluids form part of investigation in bioavailability, biochemistry drug metabolism and toxicology.
●A small quantity of drug or metabolites is usually present in a large volume of blood, urine and tissue. Solvent extraction of body fluids gives rise to an extract that may contain in addition to the drug, endogenous pigments or compounds which make analytical methods subject to error unless great care is taken e.g. choice of solvent, pH of extraction and subsequent purification methods.
●The drug may occur in free form and combined as conjugates e.g. glucuronide or ethereal sulfate, both of which are polar and water soluble. ●Protein-binding of the drug may occur and this lead to poor recovery unless the protein in denatured during the extraction procedure.
A control should be run in which the drug to be determined is added to the normal body fluids to compensate for any loss and error during the process of extraction and separation.
The electronic transition taking place in the ultraviolet and visible regions is of the following types:- *A б electron, an electron present in a б molecular orbital. *A π electron, an electron present in a π molecular orbital. *An n electron, an electron present in a nonbonding orbital
Chromophore: The term chromophore was originally used to describe an unsaturated group of atoms which were thought to be essential for color e.g. NO2 is a chromphore since its presence imparts yellow color to the compound.
Bathochromic shift or red shift:- it involves the shift of absorption maximum towards longer wavelength due to the presence of certain groups such as OH or NH2 called auxochromes or by change of the solvent, e.g. decreasing the polarity of the solvent cause red shift in the n π* transitions of carbonyl compounds. Bathochromic shift is also produced if two or more chromophores are present in conjugation in a molecule e.g. ethylene shows transition at 170nm, while 1,3-butadiene two double bonds are in conjugation shows λmax at 217nm.
b) Hypsochromic shift or blue shift. It involves the shift of absorption maximum towards shorter wavelength. It may be brought about by removal of conjugation in a system or by change of solvent. For example, protanation of aniline causes a blue shift from 280 nm to 203 nm since Ph – NH3 has no lone pair of electrons on nitrogen which can conjugate with the benzene ring.
Hyperchromic effect.This effect involves an increase in the intensity of absorption. It is usually brought about by introduction of an auxochrome. For example, introduction of methyl group in position 2 of pyridine increases Єmax (λmax 257 nm) from 2750 to 3560 (λmax 262 nm) for • π π* transition.
d) Hypochromic effect. It involves a decrease in the intensity of absorption. It is brought about by groups which distort the geometry of the molecule. For example, when a methyl group is introduced in position 2 of biphenyl group hypochromic effect takes place due to distortion caused by methyl group.
e) Auxochromes. An auxochrome is a group which itself does not act as a chromophore but when attached to a chromophore it shifts the absorption maximum towards longer wavelength along with an increase in the intensity of absorption, some commonly known auxochromic groups are –OH, -NH2, -OR, -NHR and –NR2. For example, when the auxochrome –NH2 group is attached to benzene ring, its absorption changes from λmax 255 nm (Єmax = 203) to λmax 280 nm (Єmax = 1430).
All auxochromes contain one or more non-bonding pair of electrons. When an auxochrome is attached to a chromophore it helps in extending the conjugation by sharing of non-bonded pair of electrons as shown below: + CH2 = CH –NR2 ↔:CH2-CH =NR2 The extended conjugation is responsible for bathochromic effect of auxochrome.
It may be seen that chromophores are of two types:- a)Chrmophoressuch as , and which contain both π and n electrons. They can undergo π π* as well as n π* transitions. b) Chromophores such as which contain π electrons and undergo π π* transitions.
Solvent Effects and Choice of Solvent 1- It should not absorb radiations in the region under investigation. 2- It should be less polar so that it has minimum interaction with the solute molecules. The most commonly used solvent is 95% ethanol. It is transparent above 210nm. n-hexane,cyclohexane, methanol, water, and ether. Benzene, chloroform, and carbon tetrachloride cannot be used because they absorb in the range of about 240-280nm.
Woodward - Feiser rules for calculating absorption maxima:- 1- Homoannular diene means a cyclic diene which contains conjugated double bonds in the same ring e.g.
2- Heteroannular diene is acyclic diene in which double bonds in conjugation are in separated rings e.g.