Variants In Assay Profile

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Chapter: Pharmaceutical Microbiology : Microbiological (Microbial) Assays: Antibiotics-Vitamins- Amino Acids

There are several well-recognized variants in assay profile for antibiotics, vitamins, and amino acids, namely : (1) Calibration of assay, (2) Precision of assay, (3) Accuracy of assay, and (4) Evaluation of assay performance.


VARIANTS IN ASSAY PROFILE

 

There are several well-recognized variants in assay profile for antibiotics, vitamins, and amino acids, namely :

 

(1) Calibration of assay,

 

(2) Precision of assay,

 

(3) Accuracy of assay, and

 

(4) Evaluation of assay performance.

 

The various aspects of assay profile stated above shall now be treated briefly in the sections that follows :

 

1. Calibration of Assay

 

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 solu-tions** 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.

 

Note : (1) It should be absolutely important when carying 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.

(2) 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.

 

2. Precision of Assay

 

Precision refers to – ‘agreement amongst the repeated measurements’.

 

Alternatively, precision is an exact measure of reproducibility, and is duly estimated by replicat-ing a single sample a number of times thereby determining :

·        mean result (  ) ,

·        standard deviation (SD), and

·        coefficient of variation (SD/  × 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 :

·        number replicates actually needed for each calibrator, and

·        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.

 

3. Accuracy of Assay

 

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 :

·        precision of assay, and

·        specificity of assay.

 

Poor Specificity is encountered usually in the following three instances, such as :

·        samples comprising of endogenous interfering materials,

·        presence of other antibacterial agents, and

·        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.

 

Note : 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.

 

4. Evaluation of Assay Performance

 

It has been duly proved and established that while assessing the performance characteristics of an altogether newly developed assay, both intra–and inter–assay precision duly spread over the entire range of expected concentrations must be estimated precisely.

 

Important Points : These are as stated under :

 

(1) It is extremely important to check the accuracy with the help of the ‘spiked samples’ very much spread over the entire range of concentrations used in the assay.

 

(2) Assaying ‘drug substances’ in biological fluids e.g., urine, blood, serum, sputum, cerebrospinal fluid (CSF) etc.

 

(3) Samples withdrawn from individual subjects who have been duly administered with the drug either enterally or parenterally by virtue of the fact that in vitro metabolites may only be apparent in these instances.

 

(4) Such substances that might have an inherent tendency to interfere in the assay should be thoroughly checked for there possible interference either alone or in the presence of the ‘drug sub-stance’ being assayed.

 

(5) In an ideal situation, preferentially a relatively large number of samples must be assayed both by the ‘new method’ and the ‘reference method’ individually, and the subsequent results obtained may be meticulously by linear regression ; and thus the ensuing correlation coefficient of the said two methods determined.

 

(6) Routinely employed methods may be tackled with ‘internal controls’* almost in every run ; and, therefore, the laboratories that are actively engaged in the assay of clinical specimens must take part in an external quality control programme religiously.

 

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