Welcome to ePharmacology! As pharmacology deals with the interaction of drugs with living organisms, it is essential to have reliable methods for:
- Measurement of drug potency quantitatively before use (quality control measurement)
- Measuring drug effects in order to compare quantitatively the effects of different or the same drug under different circumstances
- Measurement of concentration of drugs and other active substances in the blood, that is plasma drug concentration or other body fluids(monitoring drug therapy).
The measurement of drug concentration in the Quality Control (QC) laboratory of the drug manufacturing company is not intimately related to pharmacology but it is very much helpful for monitoring drug therapy.
The measure of drug potency in quality control laboratory is done by titration, spectrophotometry, fluorimetry, chromatography (HPLC) and bioassay. The measurement of drug in pharmacology is done mainly by bioassay, immunoassay, chromatography, mass spectrometry, spectrophotometry, fluorimetry and voltammetry.
Topics that we will learn today:
- High Performance Liquid Chromatography (HPLC)
- Mass spectrometry
The titration methods include:
- Acid-base titration
- Non-aqueous titration
- iodometric titration
1. Acid base titration
The most direct method for the determination of water soluble acid is the reaction with excess standard alkali solution (sodium hydroxide, barium or potassium hydroxide) using phenolphthalein, as indicator.
Neutralization of the alkali with standard mineral acid such as H2SO4 - sulphuric acid - produces salt of the acid having pH greater than 7 as the resulting solution will be slightly alkaline. The colour of the indicator in alkaline medium will be colourless to red (pH range of the indicator 8.3 to 10.1). The number of grams of acid required to neutralize one litre of !N NaOH, 1N Ba(OH)2 may be readily calculated. A blank experiment is also carried out.
Intro to Titration
Non Aqueous Titration
2. Non-aqueous titration:
Basic drugs may be estimated by non-aqueous titration method. Accurately weighed amount of amine drug is dissolved in glacial acetic acid and titrated with 0.1 N perchloric acid in glacial acetic acid using crystal violet indicator to get the end-point (color changes from blue to emerald green). A blank titration is also performed. The potency of the basic drug is calculated by the following formula:
% Drug = V1 X N1 X M X 100 / (W X 1000)
V1= volume of perchloric acid used
N1= normality perchloric acid
M = molecular weight of the alkaline drug
W = weight of the sample
Antibiotic co-trimoxazole may be assayed by this method.
Iodometric Titration explained simply!
3. Iodometric titration
Accurately weighed amount of drug is taken into iodine flask and dissolved in water and 20 mL of potassium iodate (potassium iodide reagent) added followed by 40 mL standard 0.1N sodium thiosulfate solution. The mixture is allowed to stand for 15 to 30 minutes and the excess thiosulfate is titrated with standard 0.1 iodine solution in presence of starch as indicator to get to the end point when the solution becomes blue.
Assay of L-ascorbic acid:
% L-ascorbic acid = A X F X 8.8 X 100 / B
where A = volume of 0.1N iodine solution titrated, B = weight of sample in mg, F = factor of 0.1N iodine solution.
For assay of vitamin C, the preparation of the sample solution is slightly different but the rest of the procedure is same.
Absorption spectrophotometry is an analytical technique in which a monochromatic (light vibrating in one plane) light beam is passed through sample solution taken in a standard cell. Organic molecules absorb energy (UV range: 200 to 300 nm) from the photon (E = h where E = energy, y = frequency of radiation and h is Plank's constant) and the fraction of light absorbed depends on:
- The nature of the length of the absorbing molecule
- Concentration of the absorbing molecule and the the length of the cell path (light path) through the solution
- The wavelength of the light
For assay purposes, the drug concentration is needed to give a satisfactory absorption signal range from roughly I-10 ug/ml. On absorption of photon (energy) the electrons of the sample move to unstable higher energy state from which energy is released in the form of another photon of lower energy that is longer wavelength than the original one. The emitted photon is detected by sensitive detector of the spectrophotometer. For comparison, the absorption of the standard substance in solution at a particular wavelength is measured and using the formula the concentration of a drug is found. For example, the assay of paracetamol is obtained from the following formula:
Potency = Absorbance (sample) X Weight of standard X Potency of standard / Absorbance (Standard) X Weight of sample
= a1 X w1 X potency of standard / a2 X w2
where, weight of standard = w in gram (gm), weight of sample = w in gram (gm), absorbance of standard and sample are a1 and a2 respectively.
This method is generally much more sensitive than absorption spectrophotometry. Only a minute portion of incident light needs to be absorbed to produce a detectable fluorescence signal. For example, vitamin B2 sample is prepared according to the standard procedure, reading is recorded by photofluorometer using primary and secondary filter and formula is used to estimate the drug.
High Performance Liquid Chromatography (HPLC)
This method is the most efficient method of separation and quantitative estimation of compounds from a mixture. Nowadays the popularity of HPLC is ever increasing in drug analysis, in measurement of the plasma drug concentration due to the reliable and reproducible results obtained from a computer within a short time.
Basically HPLC is a technique of liquid chromatography (separation technique) in which the stationary phase is packed most tightly in a narrow stainless steel column and the mobile phase is forced through the packed column under high pressure (1,000 to 3,000 psi). As the particle diameter of adsorbent material (a variety of substances, the most popular one is silica gel) is 10 p.m. or less and the column packed more tightly, development of high back pressure necessitates pumping the mobile phase (suitable solvent solvent mixture) through the column.
All the components shown in the diagram are not necessary to achieve the results. The parts connected by dashed line are only used in gradient elution and the started components are optional in both the gradient or isocratic modes. The samples to be analysed are introduced into the chromatograph by a suitable injection device using a suitable syringe to the column while mobile phase (solvent) is followed by the use of a pump (mechanical or pneumatic). In the analytical column the actual separation takes place and the result is observed in a suitable detector appropriate for a particular class of compounds. The analytical results are recorded in a recorder as peak. The particular retention or relative retention times of an unknown sample can be compared with the same parameter for a known compound to identify an unknown compound. Same compound would show the same parameter.
The area under the elution peak corresponds to the concentration of a compound in the HPLC effluent. Using computer technique the exact plasma concentration of a particular drug can be found out very easily
Mass Spectrometry (MS)
Mass spectroscopy is used widely to characterize organic molecules in the following ways:
- To measure the exact molecular weight
- To measure the exact molecular formula
- To indicate within a molecule the points at which fragmentation occurs i.e. the presence of a certain structural units in organic compound. In a mass spectrometer, volatile sample is introduced into an evacuated chamber and then ionized by bombarding the sample molecule (M) with light energy electron (70 eV) to get molecular ion (M+).
M + e- (70eV) = M+ 2e-
The resulting ions are accelerated in an electric field as beam, which is reflected by supplying a transverse magnetic field. At the end of the instrument there is a narrow slit through which ion beam passes to a detector. This detector measures the intensity or the peak at mass to charge ratio (m/z). On variation of the strength of the magnetic field, beam of ions separated according to the m/z ratio sweeps across as shown in the figure the mass spectrum of methyl alcohol, H, C-OH.
In mass spectrum, the detector is very sensitive, it can detect the sample in the range of picogram (10-12gm).
For biological samples, gas chromatography (GC) column is attached to the inlet of mass spectrometer and the technique is combinedly called GCMS (gas chromatography mass spectroscopy). The purpose is to purify the sample before being subjected to mass spectrometry analysis. This gas chromatography mass spectroscopy technique is uniquely sensitive, selective but too expensive and technically complexes for routine work. Mass spectrometry provides exact chemical identification of the components of mixture but not the quantitative amount.
The name of this method comes from voltage as this method utilizes about O.5 V ag voltage pulse to a carbon fibre microelectrode exposed to the substance to be assayed. This is a special technique which may be used in vitro to obtain an immediate read-out of the local concentration of certain endogenous mediators, by means of microelectrode introduced into the tissue.
This method is another important method of drug measurement especially to measure plasma concentration of various drugs accurately. This method is superior to other chemical assay methods as they are generally too insensitive for pharmacology.
The most important immunoassay techniques are:
- Radioimmunoassay (RIA)
- Enzyme Immunoassay (EIA) also referred to as Enzyme Linked Immunosorbent Assay (ELISA).
The requirements of Radioimmunoassay are as follows:
- An antibody which binds specifically and with high affinity to the drug to be assayed
- A radioactive material for producing a radioactively labeled version of the drug to be assayed, which is a method for separating the antibody bound drug from a free drug in the solution
The antibody (binding protein), assay sample and radioactive derivative of test sample are mixed. The antibody gets bound with sample and radioactive derivative and there remains some unbound ligands (sample and radioactive derivative). The antibody-bound species are separated from unbound ligands by gel filtration and addition of activated charcoal or finely divided silicates to extract the free ligands. After separation radioactivity in free or bound fraction is counted.
The principle of this method can be exemplified to estimate hormones. For this radioiodine labelled insulin (131I- insulin) is administered intravenously to a patient who has previously received insulin therapy. Now labelled insulin gets bound to circulating antibody Ab, generally obtained from the serum of animals e.g rabbits who have been previously immunized by repeated injection of pure hormone. A set of competing reactions between radiolabeled hormone antibody and unlabelled hormone or either standard or unknown take place.
Labelled hormone (HF) + specific antibody (Ab) = (HB.Ab) + unlabelled hormone (H) = (H.Ab), where H is the radioiodine labelled hormone which may be free (F) or bound (B).
Enzyme Immunoassay (EIA) / Enzyme-linked Immunosorbent Assay (ELISA)
In this method instead of radioisotope, an enzyme complex derivative (E-X), of the sample to be assayed is used to react with antibody (Ab) and the sample to be assayed. The three components are mixed and simple photometric (usually) measurement of enzyme activities in the mixture is done without separation step as in radioimmunoassay. It is to be mentioned here that the enzymic activity E-X-Ab is less than that of E-X. This type of assay also known as EMIT (Enzyme Multiplied Immunoassay Technique) is used for routine clinical monitoring of the antiepileptic drug phenytoin. Other developments in this technique are ir paoszes
Fluorescence Polarization Immunoassay (FPIA)
A number of drugs such as cyclosporine in plasma for therapeutic monitoring purpose are measured by this method.
Bioassay is defined as the estimation of the concentration or potency of a substance by measurement of the biological activity.
It is used to:
- Measure the pharmacological activity of new or chemically undefined substances
- Measure the concentration of known substances
- Investigate the function of endogenous mediators
- Measure drug toxicity
Bioassay is nowadays hardly used for the estimation of concentration substances. This method is only used for quantitative potency measurement under the following conditions:
- When there is no simple chemical methods
- When the chemical assay method is too complex or insensitive
- When the active principle cannot be isolated easily or may undergo decomposition during isolation procedures le. peptide hormones.
Answer these questions yourself!
- Which methods are used in drug measurement in pharmacology?
- Briefly mention the underlying principle of RIA.
- What types of compounds are estimated by fluorimetric method?
- What should be the criteria of a drug for analysis by mass spectrometry?
- What is the fundamental differences between RIA and ELISA?
- What are the uses of bioassay?
- What are the main advantages of HPLC
That's all for today! Keep coming back to ePharmacology for more awesome articles on Pharmacology!