There are, in fact, three most critical and highly explicite situations, wherein the absolute neces-sity to assay the ‘antimicrobial agents’ arise.
MICROBIOLOGICAL
(MICROBIAL) ASSAYS : ANTIBIOTICS–VITAMINS– AMINO ACIDS
INTRODUCTION
There
are, in fact, three most critical and
highly explicite situations, wherein the absolute neces-sity to assay the ‘antimicrobial agents’ arise, namely :
(a) Production i.e., in the course of commercial large-scale production for estimating the ‘po-tency’
and stringent ‘quality control’,
(b) Pharmacokinetics i.e., in determining the pharmacokinetics* of a ‘drug substance’ in humans or animals,
and
(c) Antimicrobial chemotherapy i.e., for strictly managing,
controlling, and monitoring the ensuing antimicrobial
chemotherapy**.
Summararily,
the very ‘first’ situation i.e., (a) above, essentially involves the assay of relatively high
concentration of ‘pure drug
substance’ in a more or less an uncomplicated solution, for instance : buffer solution and water.
In addition to the ‘second’ and ‘third’ i.e., (b) and (c) above,
critically involve the precise and accurate measurement at relatively low concentration of the ‘drug substance’ present
in biological fluids, namely
: serum, sputum, urine, cerebrospinal fluid (CSF), gas-tric juice, nasal
secretions, vaginal discharges etc. Nevertheless, these biological fluids by
virtue of their inherent nature invariably comprise of a plethora of ‘extranaceous materials’ which may
overtly and covertly interfere with the assay
of antibiotics.
The
actual inhibition of the observed microbial
growth under stringent standardized experimen-tal parameters may be
judiciously utilized and adequately exploited for demonstrating as well as
estab-lishing the therapeutic efficacy
of antibiotics.
It is,
however, pertinent to state here that even the slightest and subtle change duly
incorporated in the design of the antibiotic molecule may not be explicitely
detected by the host of usual ‘chemical
methods’, but will be revealed by a
vivid and clear-cut change in the observed
‘antimicrobial activity’. Therefore, the so called microbiological assays do play a great useful role for ascertaining
and resolv-ing the least possible doubt(s) with respect to the change in potency of antibiotics and
their respective formulations i.e., secondary
pharmaceutical products.
The
underlying principle of microbiological
assay is an elaborated comparison of the ‘inhibi-tion of growth’ of the microbes by a measured concentration
of the antibiotics under
investigation against that produced
by the known concentrations of a ‘standard
preparation of antibiotic’ with a known activity.
In usual
practice, two ‘general methods’ are employed extensively, such as :
(a) Cylinder-plate
(or Cup-plate) Method, and
(b) Turbidimetric
(or Tube-assay) Method.
Each of
the two aforesaid methods shall now
be discussed briefly in the sections that follows :
The cylinder-plate method solely depends
upon the diffusion of the antibiotic
from a vertical cylinder via a
solidified agar layer in a Petri-dish or plate to an extent such that the
observed growth of the incorporated microorganism is prevented totally in a
zone just around the cylinder containing a solution of the ‘antibiotic’.
The turbidimetric method exclusively
depends upon the inhibition of growth of a ‘microbial
culture’ in a particular uniform
solution of the antibiotic in a fluid medium which is quite favourable and congenial to its rather rapid
growth in the absence of the ‘antibiotic’.
Conditionalities : The
various conditionalities required for the genuine assay may be designed in such a manner that the ‘mathematical model’ upon which the potency equation is entirely based can
be established to be valid in all respects.
Examples : The various typical examples are
as stated under :
(a) Parallel-Line Model — If one
happens to choose the parallel-line
model, the two log-dose-response
lines of the preparation under investigation and the standard preparation must be parallel, i.e., they should be rectilinear
over the range of doses employed in the calculation. However, these experimental parameters need to be
critically verified by the validity
tests referred to a given probability.
(b) Slope-Ratio Method : It is
also feasible to make use of other mathematical
models, for instance : the ‘slope-ratio
method’ provided that proof of
validity is adequately demonstrated.
Based on
the copious volume of evidences cited in the literatures it may be observed
that the ‘traditional antimicrobial
agents’ have been duly determined by
microbiological assay procedures. Importantly, in the recent past
significant greater awareness of the various problems of poor assay results
specificity associated with such typical examples as :
·
partially metabolized drugs,
·
presence of other antibiotics, and
·
urgent need for more rapid/reproducible/reliable
analytical techniques ;
has
appreciably gained ground and equally encouraged the judicious investigation of
a host of other fairly accurate and precise methodologies, namely :
·
Enzymatic assays,
·
Immunological assays,
·
Chromatographic assays, including :
—High
Performance Liquid Chromatography (HPLC)
—Reverse-Phase
Chromatography (RPC)
—Ion-Pair
Chromatography (IPC)
This
chapter will cover briefly the underlying principles of these aforesaid
techniques.
Related Topics
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