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 + 80 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) 0 - 2500 0 - 200 0 - 100 Analytical sensitivity (g/l) 8 0.3 0.6 Reportable range (g/l) 10 - 2500 1 - 100 Within-run precision (%CV) 1.3 - 5.0 1.8 - 6.2 3.5 - 9.5 Between-run precision (%CV) 2.6 - 7.4 4.5 - 6.4 5.1 - 12.5 Correlation 0.98  0.92 0.95 Slope 1.07  0.78 0.89 Recovery (%) 72 - 88 98 - 104 97 - 111 GUY’S & St THOMAS’ HOSPITAL NHS TRUST