1. 20l of whole blood + 80 l of water +80 l of 0.1M ZnSO4
Simultaneous Measurement of Immunosupressants by Electrospray Tandem Mass Spectrometry (TMS) A. Sankaralingam and R Swaminathan Department of Chemical Pathology,Guy’s & St Thomas’ NHS Trust, London
INTRODUCTION
Immunosuppressants are monitored frequently during the early post transplant period
Many patients are on combined therapy
We currently use separate immunoassays.
A method to measure all three common immunosuppressants simultaneously will be an advantage
Electrospray TMS is a technique with wide potential application to routine clinical analysis.
AIM
To develop a simple multi drug analysis by electrospray tandem mass spectrometry using one point calibration
Assay Procedure
20l of whole blood l of water +80 l of 0.1M ZnSO4
Mix for 5secs
200 l of Acetonitrile containing the internal standards (10 g/l Ascomycin
&25 g/l CyD)
2 Mix for 2mins
3. Centrifuge at 15000rpm for 5mins
4. Analyse supernatant
CYCLOSPORIN - Comparison of Immunoassay (EMIT) with TMS
TACROLIMUS - Comparison of Immunoassay (MEIA) with TMS
SIROLIMUS – Comparisosn between Harefield (established TMS) and Guy’s TMS
Single point calibration (Chromsystem) vs Seven point calibration (Recipe)
Single point calibration (Chromsystem) vs Six point calibration (Abbott)
Discussion
IMMUNOASSAY
 Low assay sensitivity
Prone to interference from drug metabolites and co-adminstered drugs
Large sample volume
Single drug analysis
Expensive 
LC-MS/MS
Highly specific and sensitive
No interference because of molecular weight differences and the specificity of the
precursor-product ion combination
Limit of detection is below the therapeutic range of interest
Linearity well above therapeutic range, ideal for 2hr post CyA monitoring
Analyses accomplished with minute amount of sample
Samples can be loaded and run overnight
High sample throughput
Multi drug analysis
Low running cost although initial capital cost is high
CONCLUSION
LC-MS/MS is a specific and sensitive method for the simultaneous measurement
of immunosuppressants.
Quantitation is reliable with single point calibration
Performance Characteristics
Cyclosporin
Tacrolimus
Sirolimus
Assay Linearity (g/l)
Analytical sensitivity (g/l) 8
0.3
0.6
Reportable range (g/l)
Within-run precision (%CV)
Between-run precision (%CV)
Correlation
0.98
 0.92
0.95
Slope
1.07
 0.78
0.89
Recovery (%)
GUY’S & St THOMAS’ HOSPITAL NHS TRUST