LC-MS/MS Analysis of Drugs in Whole Blood :
A unique solution for total automation and undisturbed detection
Laboratory of
BioSeparation
Irayani Berger, Rosa Morello, Karl-Siegfried Boos
Laboratory of BioSeparation, Institute of Clinical Chemistry, Medical Center of the University of Munich, Munich, Germany
Sample
Sample Pretreatment
Pretreatment :: Goal
Goal
Anticoagulated whole blood is pumped under precisely defined conditions
(i.e. heating time and temperature) through a heated stainless steel capillary
(BloodLyser®)
2
Analyte A
Gradient elution
SystemPeak(s)
Internal
standard
Injection / Mixing
Injection / Mixing
Unit
Unit
Processing
Processing
Unit
Unit
(MD-) SPE
(MD-) SPE
Unit
Unit
Heated Capillary
Comparison
Comparisonof
of SPE-packing
SPE-packingmaterials
materials::
On-line
On-lineSPE–LC–MS/MS
SPE–LC–MS/MSanalysis
analysis
LC-MS/MS
LC-MS/MS
Unit
Unit
HPD 2
Comparison
Comparison of
of SPE-packing
SPE-packingmaterials
materials ::
On-line
On-line MD–SPE–POPLC–MS/MS
MD–SPE–POPLC–MS/MS
A) LiChrospher® ADS RP-4
HPD 1
A
I) LiChrospher® ADS RP-4 – PLR Bischoff Chromatography – Oasis® MCX
MS/MS
W
6
5
This heat-shock treatment causes disintegration of cellular blood components
and generation of a new matrix named Cell-disintegrated blood (CDB) [1].
4
Ionisation yield
Solution :
Organic modifier
1) Direct injection of anticoagulated whole blood
Flow-rate
Analyte B
Monitoring
Monitoringof
ofmatrix
matrixeffects
effectsby
bypost-column
post-columninfusion
infusionexperiments
experiments
MD–SPE–POPLC–MS/MS
MD–SPE–POPLC–MS/MS platform
platform ::
Triple
Triple cartridge
cartridgemode
mode
1
Isocratic elution
100
W
POPLC
column
Ion
suppression
B
Sample
SampleClean-up
Clean-up:: Goals
Goals
1)
Solid-Phase-Extraction (SPE) of Cell-Disintegrated Blood (CDB)
2)
Integration of SPE in analysis platform (on-line SPE)
3)
Depletion of sample matrix
4 a) Extraction of target analytes (Immunosuppressants)
b) Reduction / Elimination of matrix effects
3
7
ad 1):
Fully automated cartridge exchange and valve switching module
(ACE, Spark Holland, Netherlands)
ad 2, 3): Tailor-made SPE packing for direct injection and fractionation of
1
ad 4a):
Multidimensional SPE for highly selective sample clean-up [3]
ad 4b):
Special SPE packing for highly efficient extraction of phospholipids
(PLR, Bischoff Chromatography, Germany)
F
5
6
Waste
Valve plus T-piece
Valve plus T-piece
9
Waste
3rd SPE-cartridge
1st SPE-cartridge
min
2nd SPE-cartridge
Exchange (Step
8
)
B) HySphere C2
Fractionation
Transfer
SPE (RAM)
SPE ► LC
Separation
Re-equilibration
Time
POPLC relies on a software-assisted combination of column segments
of different length and filled with different stationary phases, i.e. C18,
C18 with enhanced polar selectivity, phenyl, C30 and cyanopropyl.
POPLC is based on the theory of the “PRISMA Model” that has been
used before to optimize mobile phases in Liquid Chromatography. The
overall retention of the analyte(s) on a POPLC-column is the sum of
the retention factors on each individual stationary phase and their
length.
Optimal or best separation of given analyte(s) are caclulated or
selected and displayed.
2
4
5
7
6
8
9
10
Fractionation
Fractionation
Separation
Separation
Direct Injection
Heat-shock
Solid Phase
High
of whole blood
treatment
Extraction
performance
Detection
Detection
%
DataDataprocessing
processing
Everolimus,
Desmethoxysirolimus (IS)
25 µL CDB
Detection
Quattro Micro MS/MS, Waters, USA
Mode : ESI+
Standard-Chromatogram
Fractionation
1. SPE cartridge :
(Triple cartridge mode)
Whole
Cell Disintegrated
Blood
Blood
(CDB)
POPLC
25 µL CDB
Detection
Quattro Micro MS/MS, Waters, USA
followed by H2O / ACN (95/5, v/v) ; Flow-rate : 3 mL/min ; 3.00 min
Transfer (SPE►AC)
Mobile Phase : MeOH / 2mM NH4Ac (90/10, v/v) ; Flow-rate : 500 µL/min ; 2.00 min
Separation
LiChrospher® 100 RP 18-EC (125x2mm) , dp 5µm
Mobile Phase : MeOH / 2mM NH4Ac (75/22, v/v) ; Flow-rate : 500 µL/min ; 10.00 min
MS/MS
0
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
Mode : ESI+
Standard-Chromatogram
Sample
Solution (25 µL) of immunosuppressants and internal standards in MeOH / H2O (80/20, v/v), each 100 ng/mL)
Fractionation
1. SPE cartridge : A) LiChrospher ADS RP 4 (10 x 2 mm ID) , dp 25 µm ;
B) HySphere C2 (10 x 2 mm ID) , dp 7 µm ;
C) HySphere C8 EC – SE (10 x 2 mm ID) , dp 8 µm ;
D) Oasis HLB (10 x 2 mm ID) , dp 30 µm
A) LiChrospher ADS RP 4 (10 x 2 mm ID) , dp 25 µm ;
B) HySphere C2 (10 x 2 mm ID) , dp 7 µm ;
C) HySphere C8 EC – SE (10 x 2 mm ID) , dp 8 µm ;
D) Oasis HLB (10 x 2 mm ID) , dp 30 µm
Mobile Phase : H2O / ACN (95/5, v/v) ; Flow-rate : 270 µL/min ; 1.00 min,
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
followed by H2O / ACN (75/25, v/v) ; Flow-rate : 3 mL/min ; 3.00 min
Transfer (1.SPE►2.SPE)
MeOH ; Flow-rate : 450 µL/min ; 3.00 min
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 2 mL/min ; 3.00 min
2. SPE cartridge : PLR Bischoff Chromatography (10 x 2 mm ID) , dp 25 µm
In-line dilution with 10mM NH4formiat , pH = 2,8 ; Flow-rate 1.5 mL/min ; 3.00 min
Temperature : 40°C
Detection
min
3. SPE cartridge : Oasis MCX (10 x 1 mm ID) , dp 30 µm
Quattro Micro MS/MS, Waters, USA
Mode : ESI+
Transfer (SPE►AC)
Mobile Phase : MeOH / 2mM NH4Ac (75/25, v/v) ; Flow-rate : 500 µL/min ; 2.00 min
Separation
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
The native, anticoagulated whole blood sample is processed in-line
by a heat-shock treatment. The resulting cell-disintegrated blood
(CDB) then is subjected to multidimensional on-line SPE.
Quattro Micro MS/MS, Waters, USA
Mode : ESI+
References
References
[1] Morello R, Milojkovic J, Boos K-S (2007) Therapeutic Drug Monitoring 29:143
[2] Boos K-S, Grimm C-H (1999) TrAC Trends in Analytical Chemistry 18:175-180
[3] Georgi K, Boos K-S (2006) Chromatographia 63:523-531
Acknowledgement
Acknowledgement
Cyclosporine D (IS)
Ascomycin (IS)
chromatography
MD - SPE
Sample
Solution (25 µL) of immunosuppressants and internal standards in MeOH / H2O (80/20, v/v),
each 100 ng/mL)
Sample
min
Solution of immunosuppressants and internal standard in MeOH / H2O (80/20), v/v), each 100 ng/mL
Flow-rate : 10 µL /min
Tacrolimus,
liquid
7) Elimination of ion suppression arising shortly after system peak
9) Elution of analyte(s) shortly after system peak
Flow-rate : 10 µL /min
Sample
We developed a fully automated analysis platform for the routine
determination of immunosuppressants in whole blood.
Cyclosporine A
8) Elimination of ion suppression by late eluting, i.e. more lipophilic matrix
components
Infusion-Chromatogram Solution of immunosuppressants and internal standard in MeOH / H2O (80/20), v/v), each 100 ng/mL
Sirolimus,
Processing
Processing
Chromatography – Oasis® MCX
Standard-Chromatogram
Infusion-Chromatogram
Summary
Summary
Injection
3) Short analysis time
Mixing
Mixing
and
and
Injection
Injection
IV) Oasis HLB® – PLR Bischoff
Chromatography – Oasis® MCX
III) HySphere C8 EC-SE – PLR Bischoff
min
min
MRM of 7 Channels ES+
TIC
1.03e6
3
1
min
min
1) Insertion of
ADS RP-4-cartridge and PLR-cartridge into clamps of
ACE-unit
2) Simultaneous conditioning of both cartridges
3) Injection of anticoagulated whole blood sample (25 µL)
4) In-line processing for 16 sec at 75°C and generation of CDB
5) Fractionation of CDB on LiChrospher® ADS RP-4-cartridge
6) Further fractionation on LiChrospher® ADS RP-4-cartridge
7) Transfer of analyte(s) from LiChrospher® ADS RP-4-cartridge onto PLR-cartridge
and extraction of phospholipids
8) Exchange LiChrospher® ADS RP-4-cartridge for PLR-cartridge and insertion of
Oasis® MCX-cartridge
9) Transfer of phospholipid-depleted fraction and analyte(s) from PLR-cartridge onto
Oasis® MCX-cartridge. Depletion of residual matrix
10) Transfer of analyte(s) from Oasis® MCX-cartridge to POPLC-column and
separation
2) Flow-rate compatible with MS/MS detection
6) Base-line separation of analyte / metabolite and isobaric internal standard
(e.g. Cyclosporine A and D)
II) HySphere C2 – PLR Bischoff
Chromatography – Oasis® MCX
Standard-Chromatogram
LiChrospher®
Set-up
Set-upof
ofTotal
TotalAnalysis
Analysis System
System (TAS)
(TAS)
1) Isocratic elution mode
5) Sufficient gap between ion suppression and elution of analyte(s)
min
D) Oasis® HLB
Fully
Fullyautomated
automatedon-line
on-lineMD–SPE–POPLC–MS/MS
MD–SPE–POPLC–MS/MSanalysis
analysisof
of
immunosuppressants
immunosuppressantsand
andInternal
InternalStandards
Standards(IS)
(IS)in
inwhole
wholeblood
blood
Analysis Sequence :
Application of Phase-Optimized Liquid Chromatography (POPLC).
Analytical
AnalyticalSeparation
Separationand
andDetection
Detection:: Goals
Goals
4) High ionization yield
C) HySphere C8 EC-SE
3
biological fluids [2]
(Restricted Access Material (RAM), LiChrospher® ADS RP-4,
Merck KGaA / VWR International GmbH, Germany)
4
E
2
W
HPLC Pumps
Solution :
Solutions :
D
C
8
Ion
suppression
22.00
24.00
26.00
28.00
min
Conditions see :
Section „Monitoring of matrix effects by post-column infusion experiments“
The use of three different SPE-cartridges and packings respectively
allows a highly selective extraction of immunosuppressants and a
highly efficient depletion of matrix components which otherwise would
interfere with the ionisation process.
This holds for hydrophilic compounds and especially for hydrophobic,
late eluting phospholipids.
Spark Holland B.V.
Waters Corporation
EUREKA PROJECT E!4112
BISCHOFF Chromatography