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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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 
Substance 
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 
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