A Rapid iMethodTM Test for Analysis of Four Immunosuppressants · iMethod™ Test A Rapid iMethodTM Test for Analysis of Four Immunosuppressants The following description outlines

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


Irayani Berger3

Derivatization

Extraction

Drying

Punching

Matrix-depleted (preprocessed) Samples



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


Irayani Berger4

On-line SPE of secondary blood specimens

Derivatization

Extraction

Drying

Punching


Supernatant



Evaporation


Dialysate

Precipitation

Centrifugation

Centrifugation

Blood Spots Dialysis Liquid-Liquid-Extraction


Anticoagulation

(Hemo) Lysate

Plasma / Serum

Plasma / Serum Filtrate

Inte

grat

ed

off-lineat-line

on-lineSPE

Matrix-containing(native) Samples

LC-MS/MS


Irayani Berger5

Derivatization

Extraction

Drying

Punching


Supernatant


Evaporation (Hemo)Lysate


Plasma / SerumDialysate Plasma / Serum Filtrate

Precipitation

Centrifugation

Centrifugation



Anticoagulation

Inte

grat

ed


off-lineat-line SPE


LC-MS/MSMatrix-containing(native) Samples

LC-MS/MSon-line

SPE


Irayani Berger6

Derivatization

Extraction

Drying

Punching


Supernatant


Evaporation (Hemo)Lysate


Plasma / SerumDialysate Plasma / Serum Filtrate

Precipitation

Centrifugation

Centrifugation



Anticoagulation

Inte

grat

ed


off-lineat-line SPE

in-line


LC-MS/MS

Cell-disintegrated Blood (CDB)


LC-MS/MSon-line

SPE


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


Irayani Berger8

Target analytes : Immunosuppressants / Internal standards

Cyclosporine DAscomycin

Desmethoxysirolimus

Tacrolimus Cyclosporine A

Everolimus

Sirolimus


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)


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


Irayani Berger11

Separation Detection Data-processing

MS/MS

Mixing and


SPE

Direct Injection

of whole blood

Heat-shock

treatment

Solid Phase

Extraction

High performance

liquid

chromatography


(CDB)

Total Analysis System (TAS) for fully automateddetermination of pharmaceuticals

(Single cartridge mode)

POPLC


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


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

http://www.poplc.de/index.html


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


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


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


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


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


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


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


SPE

Symbiosis Pharma

(Spark Holland)

POPLC

Peak compression on head of POPLC column Step gradient and flow-rate of mobile phase :


Methanol / water (57/43, v/v) 2.51 - 4.00 min at 350 µL/min


Irayani Berger17

Separation

POPLC


MS/MS

Mixing and


MD - SPE


(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)


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


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



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)




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


Mobile Phase : MeOH / 2mM NH4Ac (78/22, v/v) ; Flow-rate : 500 µL/min ; 15.00 min

Temperature : 60°C


9

8


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


Irayani Berger22

On-line MD–SPE–POPLC–MS/MS platform : Detection of Phospholipids (m/z 184 → 184)


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


Irayani Berger24

Separation

POPLC


MS/MS

Mixing and


MD - SPE


(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)


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


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)


Irayani Berger26

Shape Selectivity


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)


Fractionation 1. SPE cartridge : HySphere C8 EC-SE (10 x 2 mm ID) , dp 10 µm



Transfer (SPE ► SPE) MeOH ; Flow-rate : 450 µL/min ; 2.00 min,


2. SPE cartridge : PLR Bischoff Chromatography (20 x 3 mm ID) , dp 5 µm


Transfer (SPE ► AC) Mobile Phase : MeOH / 2mM NH4Ac (78/22, v/v) ; Flow-rate : 500 µL/min ; 30.00 min


MRM of all immunosuppressants and internal standards ; MRM m/z 184 ► 184


Irayani Berger28

Depletion of phospholipids from CDB

Fractionation (depletion) of phospholipids on PLR-cartridge by

valve-switching (Front-cut)

Efficacy : approx. 90 %


Irayani Berger29

On-line MD–SPE–POPLC–MS/MS platform:Triple cartridge mode



Fractionation 1. SPE cartridge : LiChrospher ADS RP 4 (10 x 2 mm ID) , dp 25 µm



Transfer (1.SPE►2.SPE) MeOH ; Flow-rate : 450 µL/min ; 3.00 m


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


Transfer (SPE►AC) Mobile Phase : MeOH / 2mM NH4Ac (75/25, v/v) ; Flow-rate : 500 µL/min ; 2.00 min


Mobile Phase : MeOH / 2mM NH4Ac (75/22, v/v) ; Flow-rate : 500 µL/min ; 10.00 min

Temperature : 60°C


Infusion chromatogramInfusion : Solution of immunosuppressants and internal standards, each 100 ng/mL in MeOH / H2O (80/20), v/v)


9

10


Irayani Berger30


Injection

HPLC pumps

MS/MS

Internal –T-piece

W

WW

HPD 2 HPD 1


1

2

3 4

5

6


A

B

C D E F

On-line MD–SPE–POPLC–MS/MS platform : Triple cartridge mode

Waste

POPL

C

colu

mn


Irayani Berger31


Injection

HPLC pumps

MS/MS

Internal –T-piece

W

WW

HPD 2 HPD 1


1

2

3 4

5

6


A

B

C D E F

On-line MD–SPE–POPLC–MS/MS platform : Triple cartridge mode

Waste

POPL

C

colu

mn


Irayani Berger32

Dr. Jelena Milojković

Melita Fleischmann

Team from

Spark Holland B.V.

EUREKA PROJECT E!4112

An intergovernmental initiative supporting European innovation

Waters Corporation

Bischoff Chromatography

Documents

A Rapid iMethodTM Test for Analysis of Four Immunosuppressants · iMethod™ Test A Rapid iMethodTM Test for Analysis of Four Immunosuppressants The following description outlines