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Preparation of second-line drug stock:

 

Drug preparation: Prepare the drug concentration as per national and/or internationally defined drugs critical concentration, solvent and diluents of second-line drugs for DST by MGIT.

Drugs 

Critical concentrations (μg/ml) 

Solvent  

Diluent

Kanamycin  

2.5 

DW

DW

Amikacin  

1.0 

DW

DW

Capreomycin  

2.5 

DW

DW

Ofloxacin  

2.0 

NaOH 

DW

Levofloxacin  

1.5 

NaOH 

DW

Moxifloxacin  

0.5 /1.0 

1N NaOH 

DW

Linezolid  

 1.0 

DMSO 

DW

Clofazimine  

1.0 

DMSO

DMSO

PAS 

 4.0 

Ethanol  

DW

Ethionamide

5.0

DMSO

DW

 

 

All antimicrobial agents are assayed for standard units of activity. The assay units may differ widely from the actual weight of the powder and often may differ between drug production lots. Thus, the lab must standardize the antimicrobial solutions based on the potency of an individual lot of each drug powder. 

 

The following formula is used to calculate the actual weight of the drugs to be weighed:

                    

                         Volume (mL) x Concentration (mg/mL) x dilution factor

Weight (mg) = ------------------------------------------------------------------

                                      Assay potency (mg/mg)

 

  1. Weight (milligrams) is the weight of powder needed to prepare the desired volume of stock solution at the desired concentration.

  2. Volume (in millilitres) is the desired volume of stock solution

  3. Assay potency (micrograms per milligram) is the activity or potency specified by the manufacturer of the reference standard powder. This value usually appears on the label or the certificate of analysis.

  4. Concentration (micrograms per millilitre) is the desired concentration (critical concentration) of stock solution.

  5. The dilution factor is the number of times the drug added to the tube (100ml) is getting diluted by the total volume of the medium in the tube (8.3ml) = 83

 

Stock solutions of each drug are to be made 83 times higher for MGIT. Prepare a stock solution with a higher concentration than the highest concentration required in the medium. The dilution factor for MGIT is 1:83 (7mL of MGIT media + 0.8 mL of DST supplement + 0.5 mL of test inoculum /culture).

 

Example: Amikacin

 

Concentration to be tested: 1 µg/ml

Volume of the stock solution: 20 ml

Potency of the drug: (e.g.) 716 µg /mg

Dilution factor: 83

Diluent: Sterile distilled water (DW)

Amount of drug to be weighed to prepare 20 ml of stock solution:

20 x 1 x 83

----------------- = 2.32 mg

     716

 

NOTE:

 

1. As it is difficult to weigh 2.32 mg (0.00232 grams), weigh 100 times this weight, i.e. 232 mg, and dissolve in 20 ml of sterile DW, aliquot in 500µl volume and store at – 20/ -70 oC.

 

2. At the time of setting up DST, prepare a working stock by diluting the stock 100 times, i.e. 100 µl of stock + 9.9 ml sterile DW, and add 100µl of this working solution to the designated MGIT tube.

 

3. As the assay potency is likely to vary with varying batches, care must be taken to include the correct potency factor while preparing the stock solution.

 

Resource

 

Mycobacteriology Laboratory Manual    

Assessment

 

Question​

Answer 1​

Answer 2​

Answer 3​

Answer 4​

Correct answer​

Correct explanation​

Page id​

Part of Pre-test​

Part of Post-te

Which of these solvents is NOT used for drug preparation? NaOH DMSO Ethanol PAS Answer 4 PAS is a drug and not a solvent. The solvents used for drug preparation are NaOH, DMSO, and Ethanol.      

 

 

 

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