iMethod ™ Test A Rapid iMethod TM Test for Analysis of Four Immunosuppressants The following description outlines the instrument requirements and expected results obtainable from the Spark Holland iMethod TM Test for the quantification of Cyclosporine A, Everolimus, Serolimus, and Tacrolimus when using a Spark Holland Symbiosis PICO integrated online SPE and HPLC system with a mistral column oven, an Applied Biosystems/ MDS Analytical Technologies 3200 series (API 3200™ or 3200 QTRAP ® ) LC/MS/MS instrument and Chromsystems calibrators and controls. Sample preparation is based upon precipitation of whole blood followed by centrifugation and automated clean up of the supernatant using the on-line sample preparation capabilities of the Spark PICO system when using an SPE cartridge packed with Applied Biosystems POROS ® R1 sorbent. More in depth sample preparation, and instrument parameter information is included as part of the standard operating procedure provided with the method, as is the required analytical columns. Solvents, standards and any supplies required for sample preparation are not. The mobile phase consists of the use of methanol, ammonium acetate and acetic acid with separation on a Phenomenex Luna 5 u Phenyl-Hexyl 50 x 2.1 mm HPLC column. An example chromatogram of the separation achieved is shown below in figure 1. Figure 1:Chromatogram of the level 1 Chromsystems calibrator run on an API 3200 TM LC/MS/MS System
36
Embed
A Rapid iMethodTM Test for Analysis of Four Immunosuppressants · iMethod™ Test A Rapid iMethodTM Test for Analysis of Four Immunosuppressants The following description outlines
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
iMethod™ Test
A Rapid iMethodTM Test for Analysis of Four Immunosuppressants
The following description outlines the instrument requirements and expected results obtainable from the Spark Holland iMethodTM Test for the quantification of Cyclosporine A, Everolimus, Serolimus, and Tacrolimus when using a Spark Holland Symbiosis PICO integrated online SPE and HPLC system with a mistral column oven, an Applied Biosystems/ MDS Analytical Technologies 3200 series (API 3200™ or 3200 QTRAP®) LC/MS/MS instrument and Chromsystems calibrators and controls. Sample preparation is based upon precipitation of whole blood followed by centrifugation and automated clean up of the supernatant using the on-line sample preparation capabilities of the Spark PICO system when using an SPE cartridge packed with Applied Biosystems POROS® R1 sorbent. More in depth sample preparation, and instrument parameter information is included as part of the standard operating procedure provided with the method, as is the required analytical columns. Solvents, standards and any supplies required for sample preparation are not. The mobile phase consists of the use of methanol, ammonium acetate and acetic acid with separation on a Phenomenex Luna 5 u Phenyl-Hexyl 50 x 2.1 mm HPLC column. An example chromatogram of the separation achieved is shown below in figure 1.
Figure 1:Chromatogram of the level 1 Chromsystems calibrator run on an API 3200TM LC/MS/MS System
Results The following calibration curves are representative of the performance obtained on the instrument using the method described here, and may not be representative of performance on any other instrument. Cyclosporine A Everolimus
Sirolimus Tacrolimus
Table 1: Representative calibration curves for the four immunosuppressants included in the method are shown above. The concentration for each calibrator level is summarized in the table below.
Analyte Concentration in Calibrator (ng/mL) Analyte Level
A level 0, blank, containing zero concentration of each of the four analytes was also used. Concentrations of Calibrators will vary slightly from lot-to-lot.
Analyte S/N* %CV @ Level 1 Calibrator
Cyclosporine A 118.1 1.2 Everolimus 21.2 13.0 Sirolimus 7.8 10.0
Tacrolimus 19.6 9.8 Table 3: Representative Signal to Noise Ratios * Signal to Noise (S/N) is the peak height divided by the noise measured at three standard deviations of the noise. Please note that the results presented above were obtained using a single instrument and single set of standards and samples. Prior to production use, the method should be fully validated with real samples, and the results here may not be typical for all instruments. Variations in LC column properties, chemicals, environment, instrument performance and sample preparation procedures will impact performance, thus these results should be considered as informative rather than representative. System Requirements
In order to run this method as outlined above, the following equipment and reagents are required:
• An Applied Biosystems/ MDS Analytical Technologies 3200 Series LC/MS/MS System • Spark Holland Symbiosis PICO Online SPE HPLC system with a Mistral column oven • Immunosuppressant calibrators and controls (www.chromsystems.de) • Immunosuppressant internal standards (www.sigmaaldrich.com) • LC/MS grade methanol, and ammonium acetate • A Phenomenex Luna 5u Phenyl-Hexyl 50 x 2.1 mm HPLC column • Spark SPE cartridges filled with Applied Biosystems POROS® R1 sorbent • Pipettes and standard laboratory glassware Please note that the Phenomenex HPLC is required but not included with this iMethod™ Test.
Laboratory of BioSeparation Institute of Clinical Chemistry Medical Center of the University of MunichMunich, Germany
Spark Holland Symbiosis® User Group Meeting18th September 2009, Utrecht
Multidimensional SPE and on-line POPLC-MS/MS for the analysis of immunosuppressants
in whole blood
Laboratory ofBioSeparation
Irayani Berger2
Processing of Whole Blood
Manual (off-line) / Robotic (at-line)
LC-MS/MS
Blood Spots
Laboratory ofBioSeparation
Irayani Berger3
Derivatization
Extraction
Drying
Punching
Matrix-depleted (preprocessed) Samples
Processing of Whole Blood
Manual (off-line) / Robotic (at-line)
Evaporation
Supernatant
(Hemo)Lysate
Precipitation Precipitation
Plasma / Serum
Precipitation
Plasma / Serum Filtrate
Centrifugation
Centrifugation
Dialysate
LC-MS/MS
Blood Spots Dialysis Liquid-Liquid-
Extraction
Precipitation (Hemo)Lysis MembraneFiltration
± Anticoagulation
off-lineat-lineon-line
SPE
Laboratory ofBioSeparation
Irayani Berger4
On-line SPE of secondary blood specimens
Derivatization
Extraction
Drying
Punching
Matrix-depleted (preprocessed) Samples
Supernatant
Processing of Whole Blood
Manual (off-line) / Robotic (at-line)
Evaporation
Precipitation Precipitation
Dialysate
Precipitation
Centrifugation
Centrifugation
Blood Spots Dialysis Liquid-Liquid-Extraction
Precipitation (Hemo)Lysis MembraneFiltration
Anticoagulation
(Hemo) Lysate
Plasma / Serum
Plasma / Serum Filtrate
Inte
grat
ed
off-lineat-line
on-lineSPE
Matrix-containing(native) Samples
LC-MS/MS
Laboratory ofBioSeparation
Irayani Berger5
Derivatization
Extraction
Drying
Punching
Matrix-depleted (preprocessed) Samples
Supernatant
Manual (off-line) / Robotic (at-line)
Evaporation (Hemo)Lysate
Precipitation Precipitation
Plasma / SerumDialysate Plasma / Serum Filtrate
Precipitation
Centrifugation
Centrifugation
Blood Spots Dialysis Liquid-Liquid-Extraction
Precipitation (Hemo)Lysis MembraneFiltration
Anticoagulation
Inte
grat
ed
Processing of Whole Blood
off-lineat-line SPE
Matrix-containing(native) Samples
LC-MS/MSMatrix-containing(native) Samples
LC-MS/MSon-line
SPE
Laboratory ofBioSeparation
Irayani Berger6
Derivatization
Extraction
Drying
Punching
Matrix-depleted (preprocessed) Samples
Supernatant
Manual (off-line) / Robotic (at-line)
Evaporation (Hemo)Lysate
Precipitation Precipitation
Plasma / SerumDialysate Plasma / Serum Filtrate
Precipitation
Centrifugation
Centrifugation
Blood Spots Dialysis Liquid-Liquid-Extraction
Precipitation (Hemo)Lysis MembraneFiltration
Anticoagulation
Inte
grat
ed
Processing of Whole Blood
off-lineat-line SPE
in-line
Matrix-containing(native) Samples
LC-MS/MS
Cell-disintegrated Blood (CDB)
Matrix-containing(native) Samples
LC-MS/MSon-line
SPE
Laboratory ofBioSeparation
Irayani Berger7
In-line Processing :Heat shock-treatment of an anticoagulated whole blood sample (e.g. 25 µL) under defined conditions (heating time and temperature, e.g. 16 sec at 75°C) while being pumped through a stainless-steel capillary (e.g. 300 x 0.5 mm I.D.)
Conversion of Whole Blood into Cell-Disintegrated Blood (CDB)
CDB :Homogenous, red-coloured blood specimen containing the complete matrix but no cellular components which sediment