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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
1 Level 2 Level 3 Level 4 Level 5 Level 6
Cyclosporine A 47.1 116.0 266.0 471.0 721.0 1743.0 Tacrolimus 2.3 6.3 11.4 17.7 24.6 44.0 Sirolimus 2.4 6.7 12.4 19.7 29.0 48.2
Everolimus 2.2 6.1 11.7 18.1 24.2 44.1 Table 2: Chromsystems Whole Blood Calibrator Concentrations (batch: Lot No. 2907)
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.
Important Note The purchase and use of certain of the chemicals listed above 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. Applied Biosystems 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. Legal Acknowledgements / Disclaimers The iMethod™ Test described above has been designed by Applied Biosystems 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. For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of either Life Technologies Corporation, MDS Inc., Applied Biosystems/MDS Analytical Technologies or otherwise, their respective owners. © 2009 Applied Biosystems LLC and MDS Inc. Joint Owners. All Rights Reserved. Publication no. 114AP104-01
Irayani BergerRosa MorelloKarl-Siegfried Boos
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
Whole blood Sedimented Whole blood CDB
Laboratory ofBioSeparation
Irayani Berger8
Target analytes : Immunosuppressants / Internal standards
Cyclosporine DAscomycin
Desmethoxysirolimus
Tacrolimus Cyclosporine A
Everolimus
Sirolimus
Laboratory ofBioSeparation
Irayani Berger9
Separation
HPLC
Detection Data-processing
MS/MS
Mixing and
InjectionProcessing Fractionation
SPE
Direct Injection
of whole blood
Heat-shock
treatment
Solid Phase
Extraction
High performance
liquid
chromatography
Whole blood Cell disintegrated blood
(CDB)
Total Analysis System (TAS) for fully automateddetermination of pharmaceuticals
(Single cartridge mode)
Laboratory ofBioSeparation
Irayani Berger10
Goals for the on-line SPE–LC–MS/MS analysis of immunosuppressants in whole blood
Gradient elution
Isocratic elution
System-Peak(s)
Analyte A
Analyte B
Separation Re-equilibrationTransferFractionation
SPE (RAM) SPE ► LC
Internal standard
1
2
67
Time
Org
anic
mod
ifier
Flow-rate
Ion suppression
Ioni
satio
n yi
eld
3
5
4
Laboratory ofBioSeparation
Irayani Berger11
Separation Detection Data-processing
MS/MS
Mixing and
InjectionProcessing Fractionation
SPE
Direct Injection
of whole blood
Heat-shock
treatment
Solid Phase
Extraction
High performance
liquid
chromatography
Whole blood Cell disintegrated blood
(CDB)
Total Analysis System (TAS) for fully automateddetermination of pharmaceuticals
(Single cartridge mode)
POPLC
Laboratory ofBioSeparation
Irayani Berger12
POPLC Theory
ktotal = ФAkA + ФBkB + ФCkC + … = ΣФiki
ktotal = total retention factor for an analyte of interest
ki = corresponding retention factors concerning the individual stationary phases
Фi = fraction of length of each column segment relative to the total column system
ΣФi = total column system = ФA + ФB + Фc = 1
xkA x=ktotal
Laboratory ofBioSeparation
Irayani Berger13
POPLC Theory
ktotal = ФAkA + ФBkB + ФCkC + … = ΣФiki
ktotal = total retention factor for an analyte of interest
ki = corresponding retention factors concerning the individual stationary phases
Фi = fraction of length of each column segment relative to the total column system
ΣФi = total column system = ФA + ФB + Фc = 1
Laboratory ofBioSeparation
Irayani Berger14
Determination of Rt of target analytes :Operational procedure
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.0010.0011.0012.0013.0014.00
%
1
MRM of 7 Channels ES+ TIC
2.75e53.86
3.10
5.79
6.80
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.0010.0011.0012.0013.0014.00
%
1
MRM of 7 Channels ES+ TIC
3.26e53.86
3.19 5.84
4.41 6.72
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.0010.0011.0012.0013.0014.00
%
1
MRM of 7 Channels ES+ TIC
4.95e51.84
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.0010.0011.0012.0013.0014.00
%
1
MRM of 7 Channels ES+ TIC
3.00e53.94
3.105.42
6.09
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.0010.0011.0012.0013.0014.00
%
1
MRM of 7 Channels ES+ TIC
3.05e5
5.25
4.15 10.04
6.13 12.65
ProntoSIL 100-5-C18 SH 2 (red)
ProntoSIL 100-5-Phenyl 2 (white)
ProntoSIL 100-5-CN 2 (blue)
ProntoSIL 200-5-C30 (yellow)
ProntoSIL 100-5-C18 EPS (green)
1. Determination of Rt for… : 2. Different predicted chromatograms :
POPLC column : C18 (20 mm x 3 mm ID) + C30 (20 mm x 3 mm ID) + CN (30 mm x 3 mm ID) + Phenyl (10 mm x 3 mm ID) , dp 5 µm
30 minutes
too long
3 minutes
short, but no separation of Cyclosporine A and Cyclosporine D
“Compromise” :
6 minutes
base-line separation of Cyclosporine A and Cyclosporine D
Laboratory ofBioSeparation
Irayani Berger15
9
8
7
Sample : Solution of immunosuppressants and internal standards, (each 100 ng/mL), MeOH / H2O (80/20, v/v)
Injection volume : 5 µL
Fractionation SPE cartridge : Oasis HLB (10 x 2 mm ID) , dp 25-35 µm
Mobile Phase : H2O / ACN (95/5, v/v) ; Flow-rate : 270 µL/min ; 1.85 min
Transfer In-line dilution with water ; Flow-rate 100 μL/min ; 1.85 min
Step-gradient : MeOH / 2mM NH4Ac (85/15, v/v) ; Flow-rate : 250 µL/min ; 0.00 – 6.00 min
Separation Original POPLC column : C18 (20 mm x 3 mm ID) + C30 (20 mm x 3 mm ID) + CN (30 mm x 3 mm ID) + Phenyl (10 mm x 3 mm ID) , dp 5 µm
Mobile phase : MeOH / 2 mM NH4Ac (80/20, v/v) ;
Final POPLC column : C30 (200 mm x 3 mm ID) + CN (90 mm x 3 mm ID) , dp 5 µm
Step gradient : 0.00 – 12.00 min ; MeOH / 2 mM NH4Ac (80/20, v/v) ;
12.01 – 30.00 min ; MeOH / 2 mM NH4Ac (90/10, v/v) ; Flow-rate : 250 µL/min ; Temperature : 60°C
Detection Quattro Micro, Waters, USA Mode : ESI+
5
4
2
1
On-line SPE–POPLC–MS/MS : Ion Suppression
3
Infusion chromatogramInfusion : Solution of immunosuppressants and internal standards,
each 10 ng/mL in MeOH / H2O (80/20), v/v)
Injection : 25 µL CDB
Ion Suppression
OriginalPOPLC column
Final POPLC column
Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
%
-100
0
100
Transfer and In-line dilution
6
Ascomycin / Tacrolimus
Sirolimus / Everolimus / Desmethoxysirolimus
Cyclosporine A
Cyclosporine D
Laboratory ofBioSeparation
Irayani Berger16
In-line dilution water (100 vol.%) ; 0.00 - 4.00 min at 100 µL/min
In-line dilution during transfer step
Elution of analytes from SPE-cartridge Step gradient and flow-rate of mobile phase :
Methanol / water (85/15, v/v) 0.00 – 2.50 min at 250 µL/min
Methanol / water (80/20, v/v) 2.51 – 4.00 min at 250 µL/min
SPE
Symbiosis Pharma
(Spark Holland)
POPLC
Peak compression on head of POPLC column Step gradient and flow-rate of mobile phase :
Methanol / water (61/39, v/v) 0.00 – 2.50 min at 350 µL/min
Methanol / water (57/43, v/v) 2.51 - 4.00 min at 350 µL/min
Laboratory ofBioSeparation
Irayani Berger17
Separation
POPLC
Detection Data-processing
MS/MS
Mixing and
InjectionProcessing Fractionation
MD - SPE
Whole blood Cell disintegrated blood
(CDB)
Total Analysis System (TAS) for fully automated determination of pharmaceuticals
Direct Injection
of whole blood
Heat-shock
treatment
Solid Phase
Extraction
High performance
liquid
chromatography
(Dual cartridge mode)
Laboratory ofBioSeparation
Irayani Berger18
Injection
HPLC Pumps
MS/MS
Internal –T-piece
W
1. SPE-cartridge 2. SPE-cartridge
WW
HPD 2 HPD 1
WasteInternal –T-piece
1
2
3 4
5
6
Heated CapillaryProcessing
A
B
C D E F
On-line MD–SPE–POPLC–MS/MS platform : Dual cartridge mode
Waste
POPL
C
colu
mn
Laboratory ofBioSeparation
Irayani Berger19
Elimination of ion suppression
A) Early eluting (hydrophilic) matrix components
Three-dimensional (3D) SPE :
RP + SEC + hydrophobic IEX
LiChrospher® ADS RP4 + Oasis® MCX
(Dual cartridge mode)
Laboratory ofBioSeparation
Irayani Berger20
On-line MD-SPE–POPLC–MS/MS platform: Dual cartridge mode
Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
%
0
Infusion chromatogramInfusion : Solution of immunosuppressants and internal standards,
each 100 ng/mL in MeOH / H2O (80/20), v/v)
Injection : 25 µL CDB
Sample : Solution of immunosuppressants and internal standards, (each 100 ng/mL), MeOH / H2O (80/20, v/v)
Injection volume : 25 µL
Fractionation 1. SPE cartridge : Oasis HLB (10 x 2 mm ID) , dp 25-35 µm
Mobile Phase : H2O / ACN (95/5, v/v) ; Flow-rate : 270 µL/min ; 1.00 min,
followed by H2O / ACN (95/5, v/v) ; Flow-rate : 3 mL/min ; 3.00 min
Transfer (SPE ► SPE) MeOH ; Flow-rate : 450 µL/min ; 2.00 min,
In-line dilution with 10mM NH4formiat , pH = 2,8 ; Flow-rate 2 mL/min ; 2.00 min
2. SPE cartridge : Oasis MCX (10 x 1 mm ID) , dp 25-35 µm
Mobile Phase : MeOH / 10mM NH4formiat (18/82, v/v) ; Flow-rate : 2.45 mL/min ; 2.00 min
Transfer (SPE ► AC) Mobile Phase : MeOH / 2mM NH4Ac (78/22, v/v) ; Flow-rate : 250 µL/min ; 2.00 min
Separation Original POPLC column : C18 (20 mm x 3 mm ID) + C30 (20 mm x 3 mm ID) + CN (30 mm x 3 mm ID) + Phenyl (10 mm x 3 mm ID) , dp 5 µm
Mobile Phase : MeOH / 2mM NH4Ac (78/22, v/v) ; Flow-rate : 500 µL/min ; 15.00 min
Temperature : 60°C
Detection Quattro Micro, Waters, USA Mode : ESI+
9
8
Laboratory ofBioSeparation
Irayani Berger21
On-line MD-SPE–POPLC–MS/MS platform:Comparison of matrix effects
SPE : Single cartridge mode MD – SPE : Dual cartridge modeMRM of 7 Channels ES+
TIC
6.31 e6
MRM of 7 Channels ES+
TIC
6.80 e6
(Phospho-)lipids ?
(3D)(1D)
HySphere C8 EC-SE HySphere C8 EC-SE / Oasis MCX
CyA
CyD
Asco
Tacro
Eve
Sir
Desm
CyAAsco
Tacro
CyD
Eve
Sir
Desm
Laboratory ofBioSeparation
Irayani Berger22
On-line MD–SPE–POPLC–MS/MS platform : Detection of Phospholipids (m/z 184 → 184)
Laboratory ofBioSeparation
Irayani Berger23
4
Goals for the On-line MD–SPE–POPLC–MS/MS analysis of immunosuppressants in whole blood
Gradient elution
Isocratic elution
System-Peak(s)
Analyte A
Analyte B
Separation Re-equilibrationTransferFractionation
SPE (RAM) SPE ► LC
Internal standard
1
67
Time
Org
anic
mod
ifier
Flow-rate
2
Ion suppression
Ioni
satio
n yi
eld
3
5
Ion suppression
8
9
10
Laboratory ofBioSeparation
Irayani Berger24
Separation
POPLC
Detection Data-processing
MS/MS
Mixing and
InjectionProcessing Fractionation
MD - SPE
Whole blood Cell disintegrated blood
(CDB)
Total Analysis System (TAS) for fully automated determination of pharmaceuticals
Direct Injection
of whole blood
Heat-shock
treatment
Solid Phase
Extraction
High performance
liquid
chromatography
(Triple cartridge mode)
Laboratory ofBioSeparation
Irayani Berger25
Elimination of ion suppression
A) Early eluting (hydrophilic) matrix components
Three-dimensional (3D) SPE :
RP + SEC + hydrophobic IEX
LiChrospher® ADS RP4 + Oasis® MCX
(Dual cartridge mode)
B) Late eluting (hydrophobic) matrix components
Four-dimensional (4D) SPE :
RP + SEC + shape selectivity + hydrophobic IEX
LiChrospher® ADS RP4 + PLR Bischoff Chromatography + Oasis® MCX
(Triple cartridge mode)
Laboratory ofBioSeparation
Irayani Berger27
Shape selectivity : Retention of phospholipids
Standard solution of immunosuppressants
Phospholipids in CDB
Sample : Solution of immunosuppressants and internal standards, (each 100 ng/mL), MeOH / H2O (80/20, v/v) , CDB (off-line)
Injection volume : 25 µL
Fractionation 1. SPE cartridge : HySphere C8 EC-SE (10 x 2 mm ID) , dp 10 µm
Mobile Phase : H2O / ACN (95/5, v/v) ; Flow-rate : 270 µL/min ; 1.00 min,
followed by H2O / ACN (95/5, v/v) ; Flow-rate : 3 mL/min ; 3.00 min
Transfer (SPE ► SPE) MeOH ; Flow-rate : 450 µL/min ; 2.00 min,
In-line dilution with 10mM NH4formiat , pH = 2,8 ; Flow-rate 2 mL/min ; 2.00 min
2. SPE cartridge : PLR Bischoff Chromatography (20 x 3 mm ID) , dp 5 µm
Mobile Phase : MeOH / 10mM NH4formiat (18/82, v/v) ; Flow-rate : 2.45 mL/min ; 2.00 min
Transfer (SPE ► AC) Mobile Phase : MeOH / 2mM NH4Ac (78/22, v/v) ; Flow-rate : 500 µL/min ; 30.00 min
Detection Quattro Micro, Waters, USA Mode : ESI+
MRM of all immunosuppressants and internal standards ; MRM m/z 184 ► 184
Laboratory ofBioSeparation
Irayani Berger28
Depletion of phospholipids from CDB
Fractionation (depletion) of phospholipids on PLR-cartridge by
valve-switching (Front-cut)
Efficacy : approx. 90 %
Laboratory ofBioSeparation
Irayani Berger29
On-line MD–SPE–POPLC–MS/MS platform:Triple cartridge mode
Sample : Solution of immunosuppressants and internal standards, (each 100 ng/mL), MeOH / H2O (80/20, v/v)
Injection volume : 25 µL
Fractionation 1. SPE cartridge : LiChrospher ADS RP 4 (10 x 2 mm ID) , dp 25 µm
Mobile Phase : H2O / ACN (95/5, v/v) ; Flow-rate : 270 µL/min ; 1.00 min,
followed by H2O / ACN (95/5, v/v) ; Flow-rate : 3 mL/min ; 3.00 min
Transfer (1.SPE►2.SPE) MeOH ; Flow-rate : 450 µL/min ; 3.00 m
In-line dilution with 10mM NH4formiat , pH = 2,8 ; Flow-rate 2 mL/min ; 3.00 min
2. SPE cartridge : PLR Bischoff Chromatography (10 x 2 mm ID) , dp 25 µm
Transfer (2.SPE►3.SPE) MeOH/10mM NH4formiat , pH = 2,8 (75/25, v/v) ; Flow-rate : 500 µL/min ; 3.00 min,
In-line dilution with 10mM NH4formiat , pH = 2,8 ; Flow-rate 1.5 mL/min ; 3.00 min
3. SPE cartridge : Oasis MCX (10 x 1 mm ID) , dp 30 µm
Mobile Phase : MeOH / 10mM NH4formiat (18/82, v/v) ; Flow-rate : 2.0 mL/min ; 2.00 min
Transfer (SPE►AC) Mobile Phase : MeOH / 2mM NH4Ac (75/25, v/v) ; Flow-rate : 500 µL/min ; 2.00 min
Separation Original POPLC column : C18 (30 mm x 3 mm ID) + C30 (20 mm x 3 mm ID) + CN (30 mm x 3 mm ID) + Phenyl (10 mm x 3 mm ID) , dp 5 µm
Mobile Phase : MeOH / 2mM NH4Ac (75/22, v/v) ; Flow-rate : 500 µL/min ; 10.00 min
Temperature : 60°C
Detection Quattro Micro, Waters, USA Mode : ESI+
Infusion chromatogramInfusion : Solution of immunosuppressants and internal standards, each 100 ng/mL in MeOH / H2O (80/20), v/v)
Injection : 25 µL CDB
9
10
Laboratory ofBioSeparation
Irayani Berger30
1. SPE-cartridge 2. SPE-cartridge
Injection
HPLC pumps
MS/MS
Internal –T-piece
W
WW
HPD 2 HPD 1
WasteInternal –T-piece
1
2
3 4
5
6
Heated CapillaryProcessing
A
B
C D E F
On-line MD–SPE–POPLC–MS/MS platform : Triple cartridge mode
Waste
POPL
C
colu
mn
Laboratory ofBioSeparation
Irayani Berger31
2. SPE-cartridge 3. SPE-cartridge
Injection
HPLC pumps
MS/MS
Internal –T-piece
W
WW
HPD 2 HPD 1
WasteInternal –T-piece
1
2
3 4
5
6
Heated CapillaryProcessing
A
B
C D E F
On-line MD–SPE–POPLC–MS/MS platform : Triple cartridge mode
Waste
POPL
C
colu
mn