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Immuno direct imethod flyer

A Rapid iMethod™ Test for the Analysis of Four Immunosuppressant Drugs Utilizing Direct Injection LC/MS/MS iMethod™ Test for Immunosuppressants Version 1.0 for Cliquid® Software
The following description outlines the instrument requirements Samples are prepared using a protein precipitation step, and expected results obtainable from the AB SCIEX iMethod™ centrifuged then injected for LC/MS/MS analysis. Cyclosporin D Test for the analysis of four immunosuppressant drugs. This and Ascomycin are used as internal standards. Complete details method has been validated for use on either the AB SCIEX 3200 regarding the instrumentation and sample preparation are included with the iMethod™ Test. Extraction procedures for plasma are also similar and included in the method document This method is based on the analysis of Cyclosporine A, Tacrolimus, Sirolimus, Everolimus, and the internal standards Cyclosporine D and Ascomycin in whole blood. The method utilizes Chromsystems Whole Blood Calibrators and Whole Blood Controls for determination of Cyclosporine A, Tacrolimus, The chromatogram below is representative of the data acquired. Sirolimus and Everolimus for a total run time of approx 2.0 minutes and a total cycle time of 3 minutes from injection to X IC of +M R M (6 p ai r s): 12 02. 8/4 25. 4 D a from S am pl e 4 4 (C a l 6) o f .w iff (T u rb o S p ra y ) Figure 1. Example chromatogram for Chromsystems level 1 calibrator and typical %CVs are shown in the table below.
Substance
%CV@ Level 1 calibrator
Table 1 shows the observed Signal-to-Noise (S/N) ratios for the MRM transitions of the Chromsystems Level 1 Calibrator,
including the %CV estimates from 6 replicate injections. * Signal to Noise (S/N) being the peak height divided by the noise
measured at 3 standard deviations of the noise.

Calibration
Cyclosprine A
Everolimus
1 0ju ne08 direc t inj ev alu ation.rdb (Cy closporine A): "Linear" R eg re ssion ("No" weighting): y = 0. 00246 x + 0.0 01 3 (r = 0.9999 ) 09june 08 direct inj evalu ation .rdb (Everolimus): "Lin ea r" Re gression ("No" weighting): y = 0.00787 x + 0 .00195 (r = 0.9997) Sirolimus
Tacrolimus
10june08 direct inj evaluation.rdb (Sirolimus): "Linear" Regression ("No" weighting): y = 0.00787 x + -0.00127 (r = 0.9997) 09june08 direct inj evaluation.rdb (Tacrolimus): "Linear" Regression ("No" weighting): y = 0.035 x + 0.0111 (r = 0.9998) Figure 2. Representative calibration curves for the four immunosuppressants included in the method are shown above. The concentration for
each calibrator level is summarized in Table 2.

Analyte Concentration in Calibrator (ng/mL)
Compound
Table 2. Chromsystems Whole Blood Calibrator Concentrations (batch: Lot No. 2907)
A level 0 – (blank level) containing zero concentration of each of cartridge kit are included with this iMethod™ Test. Also, that this the four analytes was also used. Concentrations of Calibrators method can also be run on other HPLC systems, given that they are supported for use by Cliquid® Software and the retention times are updated to reflect the configuration used. Note: Actual results obtained may vary due to variation in the LC column properties, chemicals, instrument performance, and Ordering Information
sample preparation procedures used. It is suggested that the method be fully validated prior to routine use. Product Name
Part Number
iMethod™ Test for Immunosuppressants Version 1.0 This method was evaluated using the calibrators from the Chromsystems whole blood calibrator set (batch: Lot No. 2907) (Includes iMethod™ Test for Mycophenolic Acid Version 1.0 for Cliquid® and controls from the Chromsystems whole blood controls Levels I, II, III and IV (batch: Lot No. 1408). Accuracy, %CV and S/N values for the target analytes were obtained using 6 low level replicates of the Level 1 Calibrator. Accuracies range from 97-105% and %CVs 3.2-11.5. The estimated quantitation limits for each analyte are more than sufficient to allow the analytical method to be used for therapeutic drug monitoring. Intra-assay precision (n=6) for the low concentration QC was between 2.3 and 15.4%CV and accuracies between 93-99%. System Requirements
To run this method as provided, the following equipment is • An AB SCIEX API 3200™ or 3200 QTRAP® Systems • A Shimadzu Prominence 20A LC System or an Agilent 1100 • Cliquid® Software for Routine Analysis • Whole blood calibrators and controls can from Chromsystems • Standards for the four immunosuppressants: Cyclosporin A, Everolimus, Sirolimus (Rapamycin), and Tacrolimus • Internal standards: Ascomycin and Cyclosporin D • HPLC grade water and methanol • High purity ammonium acetate, formic acid, and zinc sulfate • Phenomenex SecurityGuard guard cartridge kit (guard column • Phenomenex SecurityGuard Luna C18 cartridge column, 4 • Vortex mixer • Pipettes and standard laboratory glassware Please note that the Phenomenex SecurityGuard Luna C18 cartridge column and Phenomenex SecurityGuard guard While the information provided above outlines the instrument requirements and expected results obtainable from the AB SCIEX iMethod™ Test for the Analysis of Immunosuppressants, please note that the results obtained do require some experience with LC/MS/MS and sample preparation procedures. As such, web-based and on-site training are available to assist in the deployment of the iMethod™ Test and are recommended for inexperienced users. Please consult your local sales representative for more details. Important Note
The iMethod™ Test described above has been designed by AB SCIEX to provide the sample prep and instrument parameters required to accelerate the adoption of this method for routine testing. This method is provided for information purposes only. The performance of this method is not guaranteed due to many different potential variations, including instrument performance, tuning, and maintenance, chemical variability and procedures used, technical experience, sample matrices, and environmental conditions. It us up to the end user to make adjustments to this method to account for slight differences in equipment and/or materials from lab to lab as well as to determine and validate the performance of this method for a given instrument and sample type. Please note that a working knowledge of Analyst® Software may be required to do so. The purchase and use of certain of the chemicals listed below may require the end user to possess any necessary licenses, permits or approvals, if such are required in accordance with local laws and regulations. It is the responsibility of the end user to purchase these chemicals from a licensed supplier, if required in accordance with local laws and regulations. The suppliers and part numbers listed below are for illustrative purposes only and may or may not meet the aforementioned local requirements. AB SCIEX is not responsible for user’s compliance with any statute or regulation, or for any permit or approval required for user to implement any iMethod™ procedure. The information included in this product is intended for reference and research purposes only. AB SCIEX offers no guarantee as to the quality or suitability of this data and suitability of the information included in this (Library, database, etc.) for use with For Research Use Only. Not for use in diagnostic procedures.
2010 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.

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