There are several sophisticated analytical methods that are used most abundantly for the precise quantitative methods microbial assays, such as :
(a) High Performance Liquid Chromatography (HPLC),
(b) Reverse-Phase Chromatography (RPC), and
(c) Ion–Pair (or Paired-Ion) Chromatography,
These three chromatographic techniques shall now be discussed briefly in the sections that follows:
Read the rest of this entry »
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 :
Cylinder-Plate Method (Method-A)
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’.
Turbidimetric (or Tube-Assay) Method (Method-B)
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 favorable 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 :
1. partially metabolized drugs,
2. presence of other antibiotics, and
3. 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 :
a. Enzymatic assays,
b. Immunological assays,
c. Chromatographic assays, including :
— High Performance Liquid Chromatography (HPLC)
— Reverse-Phase Chromatography (RPC)
— Ion-Pair Chromatography (IPC)
There are several well-recognized variants in assay profile for antibiotics, vitamins, and amino acids, namely :
(a) Calibration of assay,
(b) Precision of assay,
(c) Accuracy of assay, and
(d) Evaluation of assay performance.
Irrespective of the method adopted for the microbial assay it is absolutely necessary to work out a proper calibration in case the ultimate result is necessarily expected in terms of the absolute units viz., mg.L– 1.
Calibrator Solutions — The calibrator solutions are essentially prepared either from a pure sample of the drug to be assayed or a sample of known potency.
Importantly, there are certain drug substances that are hygroscopic in nature ; and, therefore, their inherent potency may be expressed as:
(a) ‘as-is’ potency — which refers to — ‘the potency of the powder without drying’, and
(b) ‘dried potency’— which refers to — ‘the potency after drying to constant weight under specified/defined experimental parameters’.
Importantly, in as-is potency, the drug should be stored in such a manner that it may not lose or absorb water ; whereas, in dried potency the drug should always be dried first before weighing.
Thus, once an appropriate ‘standard materials’ is actually accomplished, the calibrator solutions usually covering a suitable range of concentrations should be prepared accordingly.
However, the actual number and concentration range of the collaborators shall solely depend on the specific type of assay being carried out. Likewise, the matrix wherein the calibrators are dissolved duly is also quite vital and important, unless it may be shown otherwise, must be very much akin to the respective matrix of the samples.
It should be absolutely important when carrying out the assay of drugs present in ‘serum’, due to the fact that protein-binding may invariably influence the ultimate results of microbiological assay predominantly.
No assay can give rise to fairly accurate results unless and until the suitable ‘calibrator solutions’ (i.e., calibrators) precisely prepared in an appropriate matrix.
Precision refers to – ‘agreement amongst the repeated measurements’.
Alternatively, precision is an exact measure of reproducibility, and is duly estimated by replicating a single sample a number of times thereby determining :
1. mean result (X) ,
2. standard deviation (SD), and
3. coefficient of variation (SD/ X × 100).
Intra-Assay Precision—usually refers to the precision within a single-run exclusively.
Inter-Assay Precision—normally refers to the precision between two or more runs.
Degree of Precision—required in a specific instance essentially will determine two cardinal factors, namely :
1. number replicates actually needed for each calibrator, and
2. number plus concentration range of calibrators.
Note : Importantly, the overall precision of several assays usually changes with concentration ; and therefore, must be assayed with low, medium, and high concentration samples.
Accuracy may be defined as — ‘a measure of the correctness of data as these correspond to the true value’.
Considering that the calibrator solutions were prepared correctly from the suitable ‘drug’, the resulting accuracy of a specific result shall exclusively depend upon two important aspects, namely :
1. precision of assay, and
2. specificity of assay.
Poor Specificity is encountered usually in the following three instances, such as :
1. samples comprising of endogenous interfering materials,
2. presence of other antibacterial agents, and
3. active metabolites of the ‘drug’ being assayed.
Positive Bias i.e., if the other drugs or drug metabolites are present simultaneously, accuracy of assay shall be expressed predominantly as a positive bias.
Negative Bias i.e., if there are antagonists present in an appreciable quantum, accuracy of assay will be expressed mostly as a negative bias.
In fact, inaccuracy caused due to apparent poor precision will invariably exhibit absolutely ‘no bias’, and that caused on account of either under–or over-potent calibrators will exhibit positive and negative bias respectively.
Just like inaccuracy that caused poor precision, bad judgment and bad place picking is also cause uncomfortable condition. Talking about place, if you are in Belarus, it is a good judgment place of apartments for rent in Minsk.
Application of pharmaceutical principles to drug dosage forms is illust rated when drug dosage forms are categorized according to their physical state, degree of heterogeneity, and chemical composition. The usual relevant states of matter are gases, liquids, and solids. Intermolecular forces of attraction are weakest in gases and strongest in solids. Conversions from one physical state to another can involve simply overcoming intermolecular forces of attraction by adding energy (heat) . Chemical composition can have a dramatic effect on physicochemical properties and behavior. For this reason, it is necessary to distinguish between polymers, or macromolecules, and more conventional ( i .e., smaller) molecules, or micromolecules.
check-tools.com
Read the rest of this entry »