Autosampler-Based Sample Preparation for Just-in-Time Extractionof Tacrolimus, Sirolimus and Cyclosporine for LC/MS Analysis

G. Lensmeyer1, A. Iwanski1, and K. Gamble21University 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 valvefor direct

connection to LC/MS

Analytical syringereplaces the probe and/or manifold of the autosampler

Tray whereITSP elutesand deposits

extracted sample

Tray withsamples

to beextracted

Storage tray with supply of ITSP SPE cartridges

Reservoirsholding wash and elution solvents

Dock where ITSP SPEcartridge

resides forconditioningand loading

sample

1. Precision StudiesThree 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 Linearity140

120

100

80

60

40

20

00 20 40 60 80 100 120 140

Conc Sirolimus Added to Whole Blood (ng/mL)

An

alyt

ical

Res

ult

(n

g/m

L) Sirolimus

n = 10r = 0.9997y = 1.00x + 0.005 ng/mLSy/x = 1.00 ng/mLLinear from 1 to at least 80 ng/mL

Cyclosporine Linearity3500

3000

2500

2000

1500

1000

500

00 500 1000 1500 2000 2500 3000 3500

Conc CsA Added to Whole Blood (ng/mL)

An

alyt

ical

Res

ult

(n

g/m

L) Cyclosporine

n = 10r = 1.0000y = 0.999x + 0.245 ng/mLSy/x = 0.512 ng/mLLinear from 25 to 2000 ng/mL

Tacrolimus Linearity80

70

60

50

40

30

20

10

00 20 40 60 80

Conc Tacrolimus Added to Whole Blood (ng/mL)

An

alyt

ical

Res

ult

(n

g/m

L) Tacrolimus

n = 10r = 0.9996y = 1.00x + 0.014 ng/mLSy/x = 0.611 ng/mLLinear from 1 to at least 80 ng/mL

4. Patient Sample Comparisons

Tacrolimus Comparisons90

80

70

60

50

40

30

20

10

00 10 20 30 40 50 60 70 80 90

Gilson Extraction Conc (ng/mL)

ITS

P E

xtra

ctio

n C

on

c (n

g/m

L)

Sirolimusn = 23r = 0.9982y = 1.08x – 1.38 ng/mLSy/x = 1.51 ng/mL

Cyclosporine Comparisons2500

2000

1500

1000

500

00 500 1000 1500 2000 2500

Gilson Extraction Conc (ng/mL)

ITS

P E

xtra

ctio

n C

on

c (n

g/m

L)

Tacrolimusn = 25r = 0.9983y = 0.972x + 0.24 ng/mLSy/x = 1.01 ng/mL

Sirolimus Comparisons100

80

60

40

20

30

10

50

70

90

020 40 60 80 100

Gilson Extraction Conc (ng/mL)

ITS

P E

xtra

ctio

n C

on

c (n

g/m

L)

Cyclosporinen = 23r = 0.9981y = 1.01x + 1.71 ng/mLSy/x = 37.6 ng/mL