DRUGS-

ASSAYS
&
TESTS

ALICIA D’SOUZA

P19004, SEM-3

PAPER-IV, PCHA304

MSc.-II
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CONTENTS

1. Assays of Enzyme containing substances ...2

2. Biological and microbiological assays and tests …5
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ENZYMATIC ASSAYS:
All colorimetric enzymatic assays essentially involve the measurement of the
activity of an enzyme under the following two circumstances, namely:
(a) When substrate is in large excess, and
(b) When enzyme concentration is in large excess.
Substrate Present in Large Excess: In reality, an enzyme reaction is nothing but
a special kind of generalized reaction that may best be expressed as follows :
Rate of Product Formation = Vmax [S]/Km + [S]
where, Km = (k2 + k3) / k1,
Assuming, [S] to be in large excess [S] >> Km,
Rate of Reaction = Vmax [S]/[S]
or Rate of Reaction = Vmax

Example: In order to measure the activity of an enzyme E, such as creatine

phosphokinase (CPK), the concentration of the substrate S, for instance
creatine, should be in large excess so that the products measured shall be in the
linear portion of the curve (Part ‘A’)
The two experimental parameters, namely: the temperature (constant-
temperature-water-bath) and the time (phaser) should always be kept constant in
order that the rate of reaction, as determined by the amount of product formed,
specially designates the activity of the enzyme under assay, and devoid of the
influence of any other variables on the reaction rate.

Enzyme Concentration in Large Excess:

In order to analyze the quantity of substrate (S) present in a biological sample
glucose oxidase is added in excess of the actual amount needed for the complete
conversion of all the substrate to product; and to achieve this object the reaction
is allowed to run for a fairly long duration (i.e., to complete the reaction). It can
be seen evident wherein the specific reaction time the substrate (S) has been
consumed completely and consequently, the concentration of the product
achieves a maximum value.
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ASSAY METHODS:
A few typical examples of colorimetric assay of enzyme levels will be discussed
briefly hereunder:

Alkaline Phosphatase (AP)

Alkaline phosphatases are a group of isoenzymes, located on the outer layer of
the cell membrane; they catalyze the hydrolysis of organic phosphate esters
present in the extracellular space. It is found to be relatively non-specific and
this characteristic permits the AP level to be assayed based on the fact that p-
nitrophenylphosphate ion gets converted to p-nitrophenolate anion at pH 10.5;
as

expressed in the following reaction.

In actual
practice, p-nitrophenylphosphate is present in large excess, and the reaction is
carried out at 38°C for 30 minutes. The resulting amount of p-nitrophenolate ion
is estimated by the help of a usual standard curve employing known
concentrations of p-nitrophenolate prepared from p-nitrophenol.

Profile of AP-levels
(1) Normal AP-levels in adults range between 0.8 to 2.3 Bessey-Lowry units
and in children between 2.8 to 6.7.
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(2) Increased AP-levels are observed in patients suffering from liver diseases,
hyperparathyroidism and in rickets.
(3) Decreased AP-levels could be seen in patients suffering from
hypoparathyroidism and pernicious anaemia (i.e., an anaemia tending to be a
fatal issue).

Lactate Dehydrogenase (LDH)

The method of LDH assay is based on kinetic analysis. In a kinetic enzymatic
assay a unit of enzyme activity is defined as ‘the quantity of enzyme that brings
about a certain absorbance increase in 30 seconds or 1 minute at a fixed
temperature (for instance 25 ± 0.2°C)’.The kinetic assay of LDH is based on the
conversion of lactic acid to pyruvic acid, in the presence of nicotinamide
adenine dinucleotide (NAD), and is closely monitored at intervals of 30 seconds
or 1 minute by measuring the increase in absorbance at 340 nm. In this
particular instance lactic acid available in an excess to ensure that the increase
in pyruvic acid is linear with time, i.e., directly proportional to time. The
reaction involved may be expressed as follows:

The liberated nicotinamide-adenine-dinucleotide hydrogenase (NADH) has an

absorption maxima at 340 nm, whereas lactic acid. NAD+ and pyruvic acid do
not absorb at all at this wavelength.
Profile of LDH-levels:
(1) Normal LDH levels are as follows :
Absorbance Units per ml : 42 to 130, International Units per ml: 0.20 to 0.063
(2) LDH level in serum is found to be increased in 8 to 10 hours after a
myocardial infarction (i.e. development or presence of an infarct in the heart);
obviously the heart muscle is destroyed and consequently the enzymes leak into
the serum.
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(3) Increased LDH levels are found in patients suffering from diseases related to
liver and renal functions, cancer and pulmonary infarction,
(4) Drugs like codeine and morphine help in enhancing LDH levels.
Procedure: The following steps need to be followed in a sequential manner :
(1) Dissolve the contents of Dermatube LDH (containing NADH and lactic
acid) with 2.8 ml of DW.
(2) Put this solution in a cuvette and then insert it in a colorimeter previously
warmed up to 25°C. Set the wavelength at 340 nm. Carefully adjust the
absorbance of this solution to 0.1 by making use of the proper variable control.
(3) Remove the cuvette and add to it 0.2 ml of serum. Mix the contents of the
cuvette and replace it quickly in position. Carefully record the absorbance
exactly at intervals of 30 seconds for 2 to 3 minutes. In case, the absorbance
happens to rise very rapidly, repeat step 3 by diluting 0.1 ml of the serum to 0.2
ml with DW.
(4) From the foregoing measurement of absorbances calculate an average
∆A/min.
(6) Report the LDH concentration as follows:

ASSAY OF SODIUM HEPARINE INJECTION:

1. This injection acts as an anti-coagulant. It is given to the patient before
operation and is determined by its ability of clotting sheep plasma invitro.
2. Assay method is compared with a reference sample and calculation of
potency is based on the extent of clotting which has occurred one hour
after the addition of sodium heparine injection.
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3. The activity of this injection is assayed on the basis of maintaining

various conditions such as pH, temperature and amount of sodium
heparine injected.
4. Seven test tubes containing uniform amount of sheep plasma with buffer
solution (CaCl2 + citric acid) are arranged and then different
concentration of Na-heparine injection is added to each tube. The volume
of injection remaining constant.
5. The extent of clotting is observed in each test tube after 1 hour at room
temperature or at 30-400 C as per pharmacopoeia. Calculation of potency
is based on the amount of Na-heparine injection which results in the
clotting of sheep plasma with level concentration.
6. Protamine sulphate injection: In this method, a dilute HCl solution of
drug is incubated with buffer solution such as ethyl ester or n-acetyl
tyrosine for 6 to 8 hours and then the change in colour is measured
spectrophotometrically at 273nm. It is the method used to determine the
amount of organic constituents which may be present in any vegetable or
plant, drug i.e. Ayurvedic medicine which has got therapeutic activity.

MICROBIAL ASSAY:
Standard and clinically isolated microorganism strains were used for
antimicrobial assays. A large number of humans, animals and plant disease are
caused by pathogenic microbes. The microbial assay for antibiotics is a method
that uses microorganisms to determine the antimicrobial potency of the
antibiotics contained in medicine.
In a cylinder plate assay, the essential comparisons are restricted to relationships
between zone diameter measurements within plates, exclusive of the variation
between plates in their preparation and subsequent handling. To conduct a
turbidimetric assay so that the difference in observed turbidity will reflect the
differences in the antibiotic concentration requires both greater uniformity in the
environment created for the tubes through closer thermostatic control of the
incubator and the avoidance of systematic bias by a random placement of
replicate tubes in separate tube racks, each rack containing one complete set of
treatments. The essential comparisons are then restricted to relationships
between the observed turbidities. Within these restrictions, two alternative
designs are recommended; i.e. a 3-level (or 2-level) factorial assay, or a 1-level
assay with a standard curve. For a factorial assay, prepare solutions of 3 or 2
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corresponding test dilutions for both the standard and the unknowns. For a 1-
level assay with a standard curve, prepare solutions of five test dilutions of the
standard and a solution of a single median test level of the unknown. Consider
an assay as preliminary if its computed potency with either design is less than
60 per cent or more than 150 per cent of that assumed in preparing the stock
solution of the unknown. In such a case, adjust its assumed potency accordingly
and repeat the assay. Microbial determinations of potency are subject to inter-
assay variables as well as intra-assay variables, so that two or more independent
assays are required for a reliable estimate of the potency of a given assay
preparation or unknown. Starting with separately prepared stock solutions and
test dilutions of both the standard and unknown. If the estimated potency of the
second assay differs significantly, as indicated by the calculated standard error,
from that of the first, conduct one or more additional assays. The combined
result of a series of smaller, independent assays spread over a number of days is
a more reliable estimate of potency than that from a single large assay with the
same total number of plates or tubes.

CYLINDER-PLATE OR CUP-PLATE METHOD:

Inoculate a previously liquefied medium appropriate to the assay with the
requisite quantity of suspension of the microorganism, add the suspension to the
medium at a temperature between 400 and 500 C and immediately pour the
inoculated medium into the petri dishes or large rectangular plates to give a
depth of 3 to 4 mm (1 to 2mm for nystatin). Ensure that the layers of medium
are uniform in thickness, by placing the dishes or plates on a level surface. Store
the prepared dishes or plates in a manner so as to ensure that no significant
growth or death of the test organism occurs before the dishes or plates are used
and that the surface of the agar layer is dry at the time of use. Using the
appropriate buffer solutions, prepare solutions of known concentrations of the
standard preparation and solutions of the corresponding assumed of
concentrations the antibiotic to be examined. Apply the solutions to the surface
of the solid medium in sterile cylinders or in cavities prepared in the agar. The
paper discs are placed on the surface of the medium. Leave the dishes or plates
standing for 1 to 4 hours at room temperature or at 40, as appropriate, as a period
of pre-incubation diffusion to minimise the effects of variation in time between
the application of the different solutions. Incubate them for about 18 hours.
Accurately measure the diameters or areas of the circular inhibition zones and
calculate the results. Selection of the assay design should be based on the
requirements stated in the individual monograph.
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Antibiotics used for this type of assay: Amphotericin B, Bacitracin, Bleomycin,

Cabenicillin, Gentamicin.

TURBIDIMETRIC OR TUBE ASSAY METHOD:

The method has the advantage of a shorter incubation period for the growth of
the test organism (usually 3 to 4 hours) but the presence of solvent residues or
other inhibitory substances affects this assay more than the cylinder plates assay
and care should be taken to ensure freedom from such substances in the final
test solutions. This method is not recommended for cloudy or turbid
preparations. Prepare five different concentrations of the standard solution for
preparing the standard curve by diluting the stock solution of the Standard
Preparation of the antibiotic and increasing stepwise in the ration 4:5. Select the
median concentration and dilute the solution of the substance being examined
(unknown) to obtain approximately this concentration. Place 1 ml of each
concentration of the standard solution and of the sample solution in each of the
tubes in duplicate. To each tube add 9 ml of nutrient medium previously seeded
with the appropriate test organism. At the same time prepare three control tubes,
one containing the inoculated culture medium (culture control), another
identical with it but treated immediately with 0.5 ml of dilute formaldehyde
solution (blank) and a third containing uninoculated culture medium. Place all
the tubes, randomly distributed or in a randomized block arrangement, in an
incubator or water-bath and maintain them at the specified temperature for 3 to
4 hours. After incubation add 0.5 ml of dilute formaldehyde solution to each
tube. Measure the growth of the test organism by determining the absorbance at
about 530 nm of each of the solutions in the tubes against the blank.
Antibiotics used for this type of assay: Amikacin, Tombramycin,
Oxytetracycline.
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REFERENCE

1. Pharmaceutical drug analysis- Ashutosh Kar Copyright © 2005, 2001,

New Age International (P) Ltd., Publishers
2. Indian pharmacopeia 2007
3. https://pharmastate.blog/bioassay-and-its-types/