Measurement of antimicrobial activity 3

Question 1

What are some situations in which antibiotic assays are likely to be performed.

Answer 1

• During the growth of the producer organism and purification of antibiotics
• During therapy with toxic antibiotics
• During product development (stability, clinical trials)
• During routine quality control (at the end of manufacturing process)

FOR THE LAST TWO, LARGE VOLUME + HIGH CONC

FOR THE FIRST ONE. LARGE VOLUME BUT LOW CONC

Question 2

What are the three methods of assaying antibiotics?

Answer 2

1) Conventional chemical assays e.g. HPLC
2) Enzyme based and immunoassays e.g. Abiotic is substrate for specific enzyme/antigen
3) Biological assays e.g. bacterial growth inhibition of test solution compared with reference standard

Question 3

Where can enzyme and immunoassay kits (rapid results) be used?

Answer 3

In hospitals e.g. for therapeutic monitoring of toxic antibiotics

Question 4

HPLC tends to be preferred in pharmaceutical industry (particularly for quality assurance applications), HOWEVER, the problem is that it may not..

Answer 4

provide a true indication

of biological activity

Question 5

Advantages of HPLC

Answer 5

• quick
• relatively accurate
• precise
• can be automated

Question 6

Disadvantages of HPLC

Answer 6

• expensive equipment
• need pure reference standards
• only one sample at a time
• the substance being assayed must be able to absorb UV light
• not always possible to resolve peaks in a sample e.g. due to excipients, degradation products

Question 7

Biological assays are used when other methods such as HPLC cannot be used for these reasons:

Answer 7

• when the antibiotic is in a solution that has a wide variety of complex substances ( that would interfere with the chemical assay e.g. serum, urine)
• when the antibiotic is present with significant concentrations of its breakdown products
• when the antibiotic has been extracted from a formulated medicine (e.g. cream, when excipients may cause interference)
• when the commercially available product is a mixture of isomers

Question 8

What are the two types of biological antibiotic assays?

Answer 8

• agar diffusion

- turbidimetric

Question 9

Both agar diffusion and turbidimetric assays permit an estimate of antibiotic _____ through direct comparison of a test antibiotic with an approved, well-calibrated, reference substance

Answer 9

potency

Question 10

What machine is used to measure turbidity?

Answer 10

Spectrophotometer

Question 11

If a biological assay is used, which type is used? Agar diffusion assay or turbidimetric assay? Why

Answer 11

Agar diffusion as it is more precise.

They are slower though.

Question 12

Advantages of bioassay

Answer 12

• cheap materials and equipment
• easy to scale up for multiple samples
• easy to perform
• measures the effect that the drug is intended to achieve (bacterial killing)
• Inactive impurities or degradation products do not interfere

Question 13

Disadvantages of bioassay

Answer 13

• slow
• imprecise
• inaccurate
• labour intensive