HPLC assay method for determining Amphotericin B levels in serum and tissue specimens

Introduction

Monitoring of serum concentrations of amphotericin B during treatment is seldom indicated. The optimum serum concentrations of the drug for particular fungal infections have not been determined. Although amphotericin B is nephrotoxic toxicity is determined by monitoring kidney function and higher blood levels do not lead to greater renal impairment.

Hazards

Normal microbiological technique is adequate for safety whilst preparing HPLC extraction of serum. Gloves should be worn whilst handling all patient samples to minimise the risk of infection. Tissue specimens must be handled inside a cabinet in a Containment Level 3 room until a suitable solvent has been added that will inactivate pathogenic organisms.

Any worker who is, or has reason to suspect that they may be pregnant should not handle acetonitrile or dimethyl sulphoxide (DMSO).

Specimens

Clotted blood specimens should be centrifuged at 3000rpm for 10 minutes and the supernatant removed for assay.

Materials

• Amphotericin B - British Pharmacopoeia Commission (Stanmore, Middlesex, U.K)
• N-acetylamphotericin B - Xechem (Newbrunswick, New Jersey, U.S.A)
• Dimethyl sulphoxide (DMSO ).
• 1% aqueous sodium dodecyl sulphate (SDS)
• Horse serum
• HPLC grade acetonitrile
• HPLC grade methanol
• 5mM - EDTA disodium salt (Ethylenediaminetetraacetic) acid

Equipment

• Gilson HPLC system - including pump, automatic sample injector and dilutor, U.V./vis wavelength detector, printer, 486 computer with Unipoint software available from Anachem, Luton, Bedfordshire, UK.
• Waters Spherisorb C8 column, 5 µm (4.6mm x 250mm) (Anachem).
• 0.5ml centrifuge tubes
• sterile pipette tips.
• Volumetric flasks
• Sterile bijou bottles
• Millipore filtration system with PTFE disposable filters (from Sartorius).

HPLC Conditions

• Mobile phase (see Appendix)
• Flow rate - 1.5ml/ min
• Injection volume - 30µl
• Run time - 10min
• Wavelength - 382nm
• Sensitivity - 0.002AUF

Quality Control

Internal Control procedure

• Control samples spiked with fixed quantities of amphotericin B are included in each HPLC extraction and run.
• Internal controls should give a value within a 10% range of the known value of amphotericin B.

Preparation of drug solutions

1. To prepare stock solutions with a concentration of 100mg/L, add 5ml of DMSO to 5mg of amphotericin B. Similarly add 5ml of DMSO to 5mg of N-acetylamphotericin B (internal standard). Then make each up to 50ml in a volumetric flask with 1% (SDS). These stock solutions can be aliquoted and stored at -70° C for at least 1 month.
2. Remove amphotericin stock solutions from the freezer, and allow them to reach room temperature.
3. When room temperature has been reached, the following amphotericin B controls should be prepared fresh in sterile horse serum on each occasion: 0.5, 1, 2, 4, 8, 16 mg/L.
4. This is achieved by adding 1.71mL of stock solution to 9ml horse serum to give 16mg/L. A series of doubling dilutions are then prepared from this concentration.
5. Prepare a 5mg/L solution of N-acetylamphotericin B in sterile distilled water by adding 0.2ml of the 100mg/L stock solution to 3.8ml of sterile distilled water. Make up fresh on each occasion.

Extraction procedure

Serum specimens

1. Add 100µl of internal standard to 100µl of each sample, control, internal control and serum blank.
2. Add 200µl of acetonitrile, mix for 30sec and let stand for 30 min.
3. Centrifuge at 10,000g for 5 min and add 100µl of the supernatant to 100µl of mobile phase. Re-centrifuge at 10,000g for 5 min and assay the supernatant.
4. A 30µl aliquot of extracted sample is then injected onto the HPLC column for analysis. Each sample takes 10 minutes to be analysed.

Tissue specimens

1. Weigh out 0.5g of tissue and add 0.5ml of internal standard (5.0mg/L).
2. Add 1ml of acetonitrile to the tissue and homogenise in a Griffiths tube. Transfer the contents of each tube into a glass quickfit tube. Vortex for 20 sec and leave to stand for 15 min.
3. Transfer to a 1.5ml centrifuge tube. Centrifuge for 10 min at 10,000g. Then transfer 200µl of each sample to a 0.5ml centrifuge tube.
4. Add 200µl of mobile phase. Vortex and re-centrifuge for 5 min at 10,000g. If a pellet remains, transfer 100µl to a new 0.5ml centrifuge tube and re-centrifuge. The sample is now ready to be analysed.
5. A 30µl aliquot of extracted sample is then injected onto the HPLC column for analysis. Each sample takes 10 min to be analysed.

Interpretations/Calculations

1. The peak area for each sample is recorded and a calibration curve for the set of standards is automatically constructed by the Unipoint software.
2. The level of amphotericin B in each of the patient's samples can then be established from this calibration curve.

Limitations

1. High concentrations of bilirubin (3mg/dL) can interfere with the HPLC of amphotericin B. Several methods have been described to overcome this problem. See Bach (1984), Granich et al. (1986), and Hosotsubo (1988).

Trouble-shooting

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

   Cause: Controls are made up incorrectly / HPLC mechanical problems

   Action required: Disregard the results and repeat the extraction with freshly made up calibration standards or if there are mechanical problems, repair these first and then re-run the samples with no re-extraction.

2. Observation: Internal control is not within 10% 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 amphotericin B obtained in mg/L for serum samples or mg/g for tissue samples.
2. The optimal serum concentrations of the drug for particular fungal infections have not been established.
3. Blood levels rise in proportion to the dose, so monitoring of blood levels during treatment is seldom indicated.

Timetable

Preparation of drug solutions:

Extraction procedure for serum:

Extraction procedure for tissue:

Interpretation / Calculation:

Appendix

Preparation of 1% SDS

1. Place 1g of SDS into a conical flask and add 100ml of sterile distilled water.

Solvent preparation

1. 5mM EDTA - Add 0.92g of EDTA disodium salt to 500ml of sterile distilled water and heat for 30min to dissolve the compound.
2. Mobile Phase - Add 300ml of ( 250ml Methanol + 175ml Acetonitrile ) to 200ml (5mM EDTA ). Filter and degas the solvent through a Millipore filtration system.

References

Mayhew JW, Fiore C, Murray T, and Barza M. An internally- standardized assay for amphotericin B in tissue and plasma. J of Chromatogr. 1983 274: 271 - 279.

Bach PR. Quantitative extraction of amphotericin B from serum and its determination by high - pressure liquid chromatography. Antimicrob. Agents Chemother 1984 Sep;26(3):314 - 317.

Granich GG, Kobayashi GS, and Krogstad DJ. Sensitive high- pressure liquid chromatographic assay for amphotericin B which incorporates as internal standard. Antimicrob. Agents and Chemother. 1986 Apr;29(4): 584 - 588.

Brassinne C, Coune A, Sculier JP, Hollaert C, Collette N and Meunier F. Chromatographic determination of amphotericin B in human serum. J of Chromatogr. 1987 419: 401 - 407.

Hosotsubo H, Takezawa J, Taenaka N, Hosotsubo K, Yoshiya I. Rapid determination of amphotericin B levels in serum by high - performance liquid chromatography without interference by bilirubin. Antimicrob. Agents and Chemother 1988 Jul;32(7): 1103 - 1105.

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