Evaluation of a rapid micro-scale assay for tacrolimus by liquid chromatography-tandem mass spectrometry.
BACKGROUND
The immunosuppressive drug tacrolimus has complex and unpredictable pharmacokinetics, therefore regular monitoring is required in patients receiving tacrolimus therapy. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring tacrolimus concentrations in whole blood and have compared it with a microparticle enzyme immunoassay.


METHODS
For the LC-MS/MS assay, samples were prepared in a 96-deep well microtitre plate by adding 10 micro L of blood to 40 micro L of 0.1 mol/L zinc sulphate solution. Proteins were precipitated by adding 100 micro L acetonitrile containing ascomycin internal standard. After vigorous mixing and centrifugation, 20 micro L of the supernatant was injected into the LC-MS/MS system. A C18 cartridge (3 mm x 4 mm) was eluted with a step gradient of 50% to 100% methanol containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid, at 0.6 mL/min. The column was maintained at 55 degrees C.


RESULTS
The retention times were 0.98 min for ascomycin and 0.98 min for tacrolimus. Cycle time was 2.5 min, injection to injection. The analytes were monitored using a Quattro micro trade mark tandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions: m/z821 > 768 (tacrolimus) and m/z809 > 756 (ascomycin). The limit of quantitation was 0.5 micro g/L and the assay was linear up to 30 micro g/L. Precision of the method, over the concentration range 2.5-15.0 micro g/L, was < 7% within-batch and < 6% between-batch. Total time to analyse 24 samples including result generation was 90 min.


CONCLUSIONS
We conclude that the LC-MS/MS method is quick, precise and robust and will provide a fast turn around of results for the transplant physician.
Keevil
B G
BG
Department of Clinical Biochemistry, Wythenshawe Hospital, South Manchester University Hospitals NHS Trust, Southmoor Rd, Manchester M23 9LT, UK. bkeevil@smuht.nwest.nhs.uk
McCann
S J
SJ
Cooper
D P
DP
Morris
M R
MR
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016903:Drug Monitoring / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood* / Q000493:pharmacokinetics; D013058:Mass Spectrometry / Q000379:methods*; D012044:Regression Analysis; D012680:Sensitivity and Specificity; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000097:blood* / Q000493:pharmacokinetics
D007166:Immunosuppressive Agents; D016559:Tacrolimus