Assay microbiological

Assay Methods. The primary assay for the streptovaricins is the microbiological assay using the agar diffusion method or a turbidimetric procedure (60). The streptovaricins can also be identified by paper (60,88) or thin-layer chromatography (3). 

As with many of the vitamins, biological assays have an important historical role and are widely used. For example, microbiological assays use l ctobacillusplantarum ATCC No. 8014 (57,59) or l ctobacillus arabinosus (60). These methods are appropriate for both nicotinamide and nicotinic acid. Selective detection of nictonic acid is possible if l euconostoc mesenteroides ATCC No. 9135 is used as the test organism (61). The use of microbiological assays have been reviewed (62). 

For most assays, the incorporated pantothenic acid has to be Hberated en2ymatically. Usually, a combination of pantotheinase and alkaline phosphatase is used to hberate the bound pantothenic acid. The official method for pantothenic acid of the Association of Official Analytical Chemists (AOAC) is the microbiological assay that uses U. Plantarium (A.TCC 8014) as the test organism (71). Samples are extracted at 121°C at pH 5.6—5.7, proteins are precipitated at pH 4.5, and the resulting clear extracts are adjusted to pH 6.8 prior to assay. This procedure is only suitable to determine calcium pantothenate or other free forms of pantothenic acid. 

The use of mutant 34486 of Neurospora crassa for the microbiological assay of ch oline has been described (8). A physiological method has also been used in which the ch oline is extracted after hydrolysis from a sample of biological material and acetylated. The acetylcholine is then assayed by a kymographic procedure, in which its effect in causing contraction of a piece of isolated rabbit intestine is measured (33). 

PCS. Partition chromatography on starch column 196, 261). MBA. Microbiological assay. 

Table XII. Microbiological Assay Methods First Used to Determine Amino Acids...

In the commonest form of microbiological assay used today, samples to be assayed are applied in some form of reservoir (porcelain cup, paper dise or well) to a thin layer of agar seeded with indicator organism. The drug diffuses into the medium and after incubation a zone of growth inhibition forms, in this case as a circle around the reservoir. All other factors being constant, the diameter of the zone of inhibition is, within limits, related to the concentration of antibiotic in the reservoir. 

These types of assay are rapid, taking approximately 2 hours, show good precision and are much more specific than microbiological assays. 

Hewitt W. Vincent S. (1989) Theory and Application of Microbiological Assay. London Academic Press. 

The potencies of some antibiotics described in the European Pharmacopoeia are determined by microbiological assay (Microbiological Assay of Antibiotics 2.7.2... 

The procedure employed for the establishment of the chemical reference substances used in these assays has been previously published (Sandrin et al. 1997). The CRSs for the microbiological assays of antibiotics are first submitted to the chemical tests of the monograph. If the results are satisfactory, a collaborative microbiological assay is carried out, using the International Standard as calibrator. Thus, these reference substances are considered to be secondary reference substances since they are calibrated against existing standards. Potency is expressed in International Units. If an International Standard does not exist, European Pharmacopoeia Units are used. 

Fig- 5-7 Results (lU/mg) of an interlaboratory study to determinate the potency of tylosin CRS 1 by microbiological assay (diffusion method). 

Fig. 5-9 Decision tree used to accept or reject results from collaborative trials to establish the potencies of antibiotics to be used as chemical reference substances for microbiological assay standards.

Exchanges between pharmacopoeias are co-ordinated by the Pharmacopoeial Discussion Group (PDG) (International Harmonisation 1995) and it is frequent that one pharmacopoeia participates in a collaborative study organized by another pharmacopoeia, or that several pharmacopoeias share the same batch of reference substance to be used in their respective monographs nevertheless, in this case the reference substance can not be considered as harmonized. A new batch of erythromycin was shared between the United States Pharmacopoeia and the European Pharmacopoeia and was established in a common coEaborative study both for the microbiological assay (used in the USP for formulations) and the liquid chromatographic assay (used in the Ph. Eur. and USP for bulk material). 

Sandrin J, Daas AGJ and Charton E (1997) Establishment of reference substances for the microbiological assay of antibiotics. Pharmeuropa 9 327-330. 

D.L. Simpson and R.K. Kobos, Potentiometric microbiological assay of gentamicin, streptomycin, and neomycin with a carbon dioxide gas-sensing electrode. Anal. Chem. 55, 1974-1977 (1983). 

The first study was performed by Venturini [97, 98] in both rats and dogs by using a microbiological assay (i.e. agar diffusion test and S. aureus 209 P FDA as test organism). Conversely from rifampicin, whose serum levels were already detectable 30 min after the administration and still measurable after 48 h, only trace amounts (i.e. 0.2 pg/ml) of rifaximin were detected in serum of fed rats 4 h later (fig. 6). The amount of detectable antibiotic was reduced by 50% in fasted animals. Similar results have been obtained in dogs after oral administration of 25 mg/ kg of both rifamycin derivatives [97, 98], No detectable amount of rifaximin was found in serum at any time. 

Good agreement with the microbiological assay is shown. However, the biggest drawbacks appear to be in silylation time and the instability of the GC column, 3 weeks at 275°C. 

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