1. Autosampler-Based Sample Preparation for Just-in-Time Extraction
of Tacrolimus, Sirolimus and Cyclosporine for LC/MS Analysis
G. Lensmeyer1, A. Iwanski1, and K. Gamble2
1University of Wisconsin Hospital & Clinics, Clinical Laboratories, Madison, WI; 2MicroLiter, Suwanee, GA
Conclusions
• The ITSP extraction platform demonstrated
excellent performance and compared well
with the proven Gilson XL4 instrument
for extracting the three immunosuppressive
drugs.
• Both devices produced data that were
analytically acceptable, however, the ITSP
stood out as offering additional benefits.
• When compared to the XL4, the ITSP
has a smaller foot print, required 60% less
reagent, 40% less sorbent mass and fewer
disposables were needed to achieve the same
performance we routinely obtain with the
XL4.
• Direct integration of the ITSP with the
LC/MS permits unattended processing
and assaying of samples which can translate
to decreased labor costs and improved
efficiency.
• Most importantly, ITSP and XL4 produce
relatively clean extracts, thereby allowing
the use of LC/MS rather than the more
costly LC/MS/MS to achieve the required
sensitivity, selectivity and freedom from ion
suppression.
References
1. ITSP by MicroLiter Analytical Supplies, Inc. is protected
by the following US Patents: 6, 859, 615 and 7, 001, 774.
Foreign patents apply.
2. Poquette M, Lensmeyer G, Doran T. Effective use
of liquid chromatography-mass spectrometry (LC/MS)
in the routine clinical laboratory for monitoring
sirolimus, tacrolimus and cyclosporine. Ther Drug Monit
2004;27(1):1-7.
Acknowledgement
We wish to thank Dr. Donald Wiebe for his support
of this project.
Introduction
• Time-consuming manual processing of solid phase extractions (SPE) can
negatively impact analytical precision and hinder efficiency.
• Automation of SPE would be ideal for high-volume tests requiring quick
turnaround of results in a clinical setting.
• We investigated a newer form of automation, the “Instrument Top Sample
Prep (ITSP)”, designed by MicroLiter Analytical Supplies and integrated on the
CTC Analytics PAL module1
. We applied our previously published2
method
of extraction for three immunosuppressive drugs, Tacrolimus (TAC), Sirolimus
(SIRO) and Cyclosporine (CSA) from whole blood to the ITSP. Liquid
chromatography/mass spectrometry (LC/MS) was used to analyze the whole
blood extracts generated by the ITSP.
• For comparison studies, we ran patient samples in tandem with our published
method that incorporates the Gilson XL4 liquid handler for SPE extractions.
The differences in the two extraction devices are that the ITSP processes one
sample at a time through the complete SPE and introduces a portion of the final
eluate directly into the LC/MS before processing the next sample. The Gilson
XL4 extracts four samples at a time and requires transfer of final eluate to the
LC/MS autosampler.
• Here, we present our method validation data for the ITSP with comparison to the
Gilson XL4 performance.
ITSP/CTC Analytics PAL
Results
Within-Run Precision (n = 20)
Lyphochek 1 Tacrolimus Sirolimus Cyclosporine
Mean (ng/mL) 3.49 4.03 73.69
Std. Dev. (ng/mL) 0.13 0.23 1.39
CV (%) 3.6 5.7 1.9
Utak 2
Mean (ng/mL) 15.07 18.09 515.5
Std. Dev. (ng/mL) 0.38 0.49 5.19
CV (%) 2.5 2.7 1.0
Utak 3
Mean (ng/mL) 22.52 28.19 1196.3
Std. Dev. (ng/mL) 0.46 1.01 13.29
CV (%) 2.1 3.6 1.1
Between-Run Precision (n = 20)
Lyphochek 1 Tacrolimus Sirolimus Cyclosporine
Mean (ng/mL) 3.63 3.86 78.22
Std. Dev. (ng/mL) 0.14 0.30 3.34
CV (%) 3.8 7.8 4.3
Utak 2
Mean (ng/mL) 15.45 18.47 526.8
Std. Dev. (ng/mL) 0.29 0.63 6.82
CV (%) 1.9 3.4 1.3
Utak 3
Mean (ng/mL) 23.56 29.85 1228.63
Std. Dev. (ng/mL) 0.58 1.15 20.02
CV (%) 2.5 3.8 1.6
Between-Run Precision for the Gilson XL4 Extraction
Lyphochek 1 Tacrolimus Sirolimus Cyclosporine
Mean (ng/mL) 3.54 3.70 72.13
Std. Dev. (ng/mL) 0.153 0.263 1.94
CV (%) 4.3 7.1 2.7
Utak 2
Mean (ng/mL) 15.11 18.59 526.8
Std. Dev. (ng/mL) 0.463 0.895 10.47
CV (%) 3.1 4.8 1.9
Utak 3
Mean (ng/mL) 23.32 30.01 1231.1
Std. Dev. (ng/mL) 0.839 1.909 28.76
CV (%) 3.6 6.3 2.33
Tacrolimus Sirolimus Cyclosporine
0.8 – 1.0 ng/mL 1.5 – 1.8 ng/mL 8 – 10 ng/mL
Methods
2A. Analytical syringe
picks up a ITSP SPE
cartridge packed with 15
mg SDBL (Phenomenex,
Torrance, CA) and carries it
to the Dock.
2E. The sample is then
forced through the SPE
cartridge.
2F. The syringe aspirates
0.5 mL of acetontrile/
water, 30/70 (v/v) from
the reservoir and forces the
wash through the ITSP
SPE cartridge.
2G. The syringe aspirates
0.6 mL of elution solvent
(acetonitrile), moves to the
Dock, picks up the ITSP
SPE cartridge and moves
the unit to the elution tray
where the solvent is forced
through the cartridge. The
eluate flows directly into a
vial.
2H. A portion (1 – 5 µL) of the extract is drawn up by the
analytical syringe, the manifold moves to the injector and
the sample is introducted into the LC/MS for analysis of the
three immunosuppressive drugs. Alternatively, the vials can
be transferred to the autosampler of the LC/MS. We choose
the latter for this study.
3. Parameters and conditions for the LC/MS analysis are
listed in Reference 1. The following chromatograms are
typical of this analysis and display results of the compounds
detected as sodium adducts [M + 23]+
.
2. Extraction Process with the ITSP:
1. Whole Blood (EDTA) proteins are precipitated and the
protein-depleted supernatant is placed on the CTC Analytics
PAL (PAL) modified for ITSP for extraction of TAC, SIRO
and CSA. Briefly, 250 µL of whole blood is combined with
750 uL of a mixture of acetonitrile/water/zinc sulfate hepta-
hydrate (350 g/L), 50/50/5 (by vol) containing the internal
standards ascomycin (5 ng/mL), desmethoxysirolimus (10
ng/mL) and cyclosporine G (75 ng/mL). After a 10 min
incubation at room temperature, the sample is centrifuged
and the supernatant is poured over into a vial containing
500 µL water. This vial is placed in the sample tray of the
PAL autosampler.
2B. The syringe aspirates
0.5 mL of acetonitrile.
2C. The acetonitrile is
forced through the ITSP
SPE cartridge to waste.
A 0.5 mL portion of the
second conditioning
solution (acetonitrile/water,
10/90 [v/v]) is applied.
2D. The syringe aspirates
1.0 mL of the diluted
protein-depleted whole
blood supernatant located
in the sample tray.
Time (minutes)
0.3 0.5 0.7 0.9 1.1
0.3 0.5 0.7 0.9 1.1
0.3 0.5 0.7 0.9 1.1
0.3 0.5 0.7 0.9 1.1
m/z 937
m/z 827
m/z 815
m/z 907
sirolimus
rt = 0.76
tacrolimus
rt = 0.76
ascomycin
rt = 0.77
desmethoxysirolimus
rt = 0.81
0.3 0.5 0.7 0.9 1.1 1.3
0.3 0.5 0.7 0.9 1.1 1.3
m/z 1225
m/z 1239
cyclosporine A
rt = 0.83
cyclosporine G
rt = 0.89
Time (minutes)
Injection valve
for direct
connection
to LC/MS
Analytical syringe
replaces the probe
and/or manifold of
the autosampler
Tray where
ITSP elutes
and deposits
extracted
sample
Tray with
samples
to be
extracted
Storage tray with
supply of ITSP
SPE cartridges
Reservoirs
holding wash and
elution solvents
Dock where
ITSP SPE
cartridge
resides for
conditioning
and loading
sample
1. Precision Studies
Three whole-blood control products (Utak 2, Utak 3, BioRad Lyphochek 1) that
contain cyclosporine, tacrolimus and sirolimus in a range of concentrations were
assayed.
2. Low Limit of Quantitation (LLQ)
3. Linearity
Sirolimus Linearity
140
120
100
80
60
40
20
0
0 20 40 60 80 100 120 140
Conc Sirolimus Added to Whole Blood (ng/mL)
AnalyticalResult(ng/mL)
Sirolimus
n = 10
r = 0.9997
y = 1.00x + 0.005 ng/mL
Sy/x = 1.00 ng/mL
Linear from 1 to at least 80 ng/mL
Cyclosporine Linearity
3500
3000
2500
2000
1500
1000
500
0
0 500 1000 1500 2000 2500 3000 3500
Conc CsA Added to Whole Blood (ng/mL)
AnalyticalResult(ng/mL)
Cyclosporine
n = 10
r = 1.0000
y = 0.999x + 0.245 ng/mL
Sy/x = 0.512 ng/mL
Linear from 25 to 2000 ng/mL
Tacrolimus Linearity
80
70
60
50
40
30
20
10
0
0 20 40 60 80
Conc Tacrolimus Added to Whole Blood (ng/mL)
AnalyticalResult(ng/mL)
Tacrolimus
n = 10
r = 0.9996
y = 1.00x + 0.014 ng/mL
Sy/x = 0.611 ng/mL
Linear from 1 to at least 80 ng/mL
4. Patient Sample Comparisons
Tacrolimus Comparisons
90
80
70
60
50
40
30
20
10
0
0 10 20 30 40 50 60 70 80 90
Gilson Extraction Conc (ng/mL)
ITSPExtractionConc(ng/mL)
Sirolimus
n = 23
r = 0.9982
y = 1.08x – 1.38 ng/mL
Sy/x = 1.51 ng/mL
Cyclosporine Comparisons
2500
2000
1500
1000
500
0
0 500 1000 1500 2000 2500
Gilson Extraction Conc (ng/mL)
ITSPExtractionConc(ng/mL)
Tacrolimus
n = 25
r = 0.9983
y = 0.972x + 0.24 ng/mL
Sy/x = 1.01 ng/mL
Sirolimus Comparisons
100
80
60
40
20
30
10
50
70
90
0
20 40 60 80 100
Gilson Extraction Conc (ng/mL)
ITSPExtractionConc(ng/mL)
Cyclosporine
n = 23
r = 0.9981
y = 1.01x + 1.71 ng/mL
Sy/x = 37.6 ng/mL