Bioassay for Determining Voriconazole Levels in Blood

Introduction

Serum concentrations of voriconazole should be measured in patients receiving this drug to ensure that therapeutic levels are being achieved. The assay will give an indication of whether suitable blood levels have been achieved.

Hazards

Normal microbiological technique is adequate for safety whilst preparing the bioassay plate. Gloves should be worn whilst handling all patient samples, and in cases of high-risk specimens, a class I safety cabinet should be used. Any worker who is, or has reason to suspect they may be, pregnant should not handle dimethylformamide.

Specimens

  • Serum - minimum volume 200µl
  • Plasma - minimum volume 200µl

Samples may also be in the form of clotted blood (with or without anticoagulant), from which plasma/serum should be obtained by spinning the samples at 3000rpm in a centrifuge for 10 minutes.

Materials

(see Appendix for preparation of media)

  • RPMI-1640 medium with L-glutamine and NaHCO3
  • Morpholinopropanesulfonic acid (MOPS)
  • Glucose
  • Base agar No. 1
  • Voriconazole - Pfizer, Sandwich, Kent
  • Pooled negative serum/plasma
  • Dimethylformamide


Equipment

  • 60ºC water bath
  • Haemocytometer
  • Plastic bioassay plate (manufactured by NUNC)
  • Cork borer No. 3 (8mm holes)
  • Template for spacing holes
  • Dial calipers
  • Semi-logarithmic graph paper
  • Levelling table
  • Spirit level
  • Digital pipettes and sterile pipette tips
  • Sterile bijou bottles

Quality Control

Internal control procedure -

1) Control samples with known amounts of voriconazole are placed on each bioassay plate.

2) Internal controls should give a value within a 20% range of the known value of voriconazole.

Procedure

1) Using a fume cupboard, whilst wearing rubber gloves, safety glasses and a dust mask, prepare a stock solution of voriconazole, at 1000mg/l, by dissolving 0.003g of voriconazole powder in 0.3ml of dimethylformamide, then adding 2.7ml of sterile distilled water. This solution should be vortexed well and dispensed into 200µl amounts, and stored at -20ºC.

2) Melt the RPMI agar (100ml) and then place in a 60ºC water bath.

3) Prepare a suspension of sensitive Candida kefyr San Antonio strain (2 large loopfuls) in 5ml of sterile distilled water. Count cells using a haemocytometer and adjust to 1 x 107 cells per ml.

4) Label orientation of bioassay plate.

5) Using a levelling table ensure the plate is completely flat.

6) Mix the cooled RPMI agar and the yeast suspension, and pour the bioassay ensuring no bubbles are present. Leave to solidify for at least 30 minutes.

7) Take the voriconazole stock solution (1000mg/l) and pooled negative serum/plasma out of the freezer, and allow to reach room temperature.

8) When room temperature has been reached, dilute the voriconazole stock solution in pooled negative serum to give a range of concentrations from 3.12mg./l to 0.098mg/l. This is achieved by diluting 100µl of the original drug stock (1000mg/l) in 900µl of serum to give 100mg/l. Doubling dilutions in 500µl amounts are then carried out in serum/plasma.

9) When the agar has solidified, dry the plate, inverted with the lid open, at 37ºC.

10) Once the agar surface has dried, cut out 36 wells of 8mm in diameter with sterile cork borer No. 3. (6 rows of 6)

11) Using the template, place 40µl of standard or patient specimen into the appropriate wells. Always test in duplicate or triplicate using a randomised pattern.

12) Allow the drug to pre-diffuse by leaving the plate to stand at room temperature for 30 minutes.

13) Incubate the plate at 37ºC overnight (approximately 18 hours).

14) Measure the diameters of the zones of inhibition around each well using dial calipers, to the nearest 0.1mm, and record on the results form.

Interpretations/Calculations

1) Calculate the mean diameter for each standard and patient sample.

2) Plot the mean diameter of standards against voriconazole drug concentrations on semi-logarithmic graph paper, with the drug concentrations on the logarithmic ordinate. Draw the best fit straight line.

3) Use the graph to estimate the concentration of drug in the patient specimens and internal standards.

Limitations

1) Almost any Candida kefyr isolate is suitable providing it is susceptible to voriconazole.

2) Care must be taken when specimen details suggest that the patient is receiving another antifungal drug in addition to voriconazole. In such cases a HPLC assay may be necessary, or a bioassay using an organism resistant to the second antifungal.

Trouble-shooting

1) Observation: A good standard curve is not obtained.

Cause: Standards are made up incorrectly/bioassay plate prepared incorrectly/plate read incorrectly.

Action required: Disregard results and repeat bioassay after a careful review of all steps in the protocol. If possible use new batches of medium, plasma/serum and antifungal drug stock.

2) Observation: Internal control is not within 20% of the expected value.

Cause: As above, or internal control has deteriorated.

Action required: Check previous internal control results for signs of deterioration. If none apparent, take action as above.

Reporting

1) Report as the value of voriconazole obtained in mg/l.

2) As voriconazole is a trial drug optimal serum concentrations have not yet been established.

Timetable

(1) 15 minutes

(2-5) 30 minutes, depending on the time taken for the agar to melt.

(6-8) 1 hour since 30 minutes required for the plate to set.

(9-12) 1 hour

(13) Overnight

(14) 30 minutes

Interpretations/calculations:

(1-3) 30 minutes

Appendix

Preparation of RPMI Agar with 2% Glucose

1) Place 900ml of RPMI-1640 medium into a conical flask.

2) Add 34.5g of morpholinopropanesulfonic acid (MOPS) and stir to dissolve.

3) Add 20g of glucose and continue stirring to completely dissolve.

4) Adjust the pH to 7.0 using 10M NaOH.

5) Make up to 1 litre with RPMI-1640 and re-check pH.

6) Place 1.5g of Base agar No. 1 and 100ml of the prepared RPMI-1640/2% glucose medium into each medical flat.

7) Sterilise in an autoclave for 15 minutes at 121ºC (15psi).

8) This agar can be stored at room temperature for up to 3 months.

 

HPLC can also be used to measure serum concentrations of voriconazole:

Gage R, Stopher DA. J Pharm Biomed Anal 1998 Sep;17(8):1449-53 A rapid HPLC assay for voriconazole in human plasma.

Gopher DA, Gage R. Determination of a new antifungal agent, voriconazole, by multidimensional high-performance liquid chromatography with direct plasma injection onto a size-exclusion column. J Chromatogr B Biomed Sci Appl 1997 Apr 11;691(2):441-8

Year prepared: 

2010