Purpose

Liquid chromatography (LC) in combi-nation with tandem mass spectrometry(MS/MS) has the potential for simulta-neous determination of Cyclosporin A,Tacrolimus, Sirolimus, and Everolimuswith greater specificity, lower detectionlimits, and a broader dynamic rangethan commercial immunoassays. Severalapproaches have been proposed for anLC/MS/MS procedure for this purpose.

For optimal speed, stability, and long-term robustness of these methods,configuration of the mass spectrometermust be given careful consideration. Inaddition, since minimal manual samplehandling and rapid sample turnaroundare highly desireable, sample prepara-tion is also of paramount importance.

Overview

This research note describes a proposedLC/MS/MS procedure for the simulta-

neous assay of Cyclosporin A,Tacrolimus (also known as FK-506),

Sirolimus (Rapamycin) and Everolimus

(RAD-001) based on a simple androbust hardware configuration. Usingthis method, the concentration andidentification of these immunosuppres-sant drugs can be accomplished quicklyand simply, with minimal manual

sample clean-up and without compro-

mising assay precision and accuracy.Results presented in this research note

demonstrate the robustness of thismethod and the significant time savingsin the sample preparation step.

Key features

• Sensitive and selective MultipleReaction Monitoring (MRM) scanfunction for linear quantitativeanalysis with wide dynamic range

• Patented LINAC® collision cellallows simultaneous multi-compo-

nent analysis at reduced MRM dwelltimes maintaining sensitivity andpreventing crosstalk. This reducessignificantly the occurrence of falsepositive results.

• Rugged and reliable triplequadrupole MS system formaximum uptime

• ANALYST® software control of all

peripheral devices for reducedsolvent consumption, highreproducibility and ease of use.

Experimental conditions

Samples were prepared by adding 200 µL of a diluting solution* to

100 µL of either blood, calibrators orcontrols, and vortex mixing for 30 seconds. After centrifugation at13,000 g x 5 min., 200 µL of super-natant were transferred to either anautosampler vial or a microtiter plate.

The hardware configuration includedan Applied Biosystems/MDS SCIEXAPI 3000™ LC/MS/MS System TripleQuadrupole Mass Spectrometerequipped with a TurboIonSpray®

source. The source operates in positiveion mode at a voltage of +5500 voltsand with a “turbo” gas flow of 8 L/minof air heated at 300° C (nominalheating-gun temperature).

Multiple Reaction Monitoring (MRM)measurements were made using declus-

tering potential (DP) and collisionenergy (CE) values which were auto-matically optimized by the software foreach of the analytes. DP values rangedfrom 50 to 60 V; CE values werebetween 20 and 50 eV for the singly

charged species measured there.

Two-dimension chromatography wasperformed through a split arrangement

of the modules of a Perkin Elmer Series200 Micro Pump and a computer-

controlled Valco Valve (10-port,

2 positions), plumbed as depicted inFigure 1. First dimension chromato-graphy was accomplished via anApplied Biosystems POROS® R1/20,2.1 x 30 mm perfusion-column.Second dimension chromatography was

performed using a Phenomenex Luna 5 µm Phenyl-Hexyl, 2 x 50 mmcolumn (p.n. 00B-4257-B0) housed in

A simple and rapid method for the simultaneousdetermination of 4 immunosuppressant drugsusing LC/MS/MS

Application Note - Clinical Research

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an oven at 60 °C. A 10 µL sample wasinjected into the column via a Perkin Elmer Series 200 Autosampler,plumbed as depicted in Figure 1. All peripheral devices and flowrates areautomatically controlled by the ANALYST® software.

All the hardware set-up details, solutionand reagents requirements, and operat-ing parameters were stored on CD for aquick transfer of the proposed method-ology.

* Diluting solution consisted of 80%

methanol at 80 % with 1% ZnSO4, and

20 ng/mL of Ascomycin and 200 ng/mL of

Cyclosporin D as internal standards

Results and discussion

Figure 2 shows the single MS and theMS/MS spectra collected via infusion of a standard solution of Sirolimus(Rapamycin). Under the optimized

experimental conditions, the ammo-nium adduction, [M+NH4]+ at m/z931.6 Da, was observed as the basepeak. There was also some evidence ofthe Na+ adduct (not shown in this figure).

Fragmentation of the m/z 931.6 precur-

sor ion resulted in an MS/MS spectrumwith base peak ion at m/z 864.4 Da.

For quantitation by MRM, thetransition 931.6 > 864.4 was chosen. The same optimization procedure was

followed for the remaining analytes(Tacrolimus, Cyclosporin A andEverolimus) and the internal standards

(Cyclosporin D and Ascomycin).

Figure 3 shows a typical trace for a

blood sample obtained using theproposed hardware configuration.Under these optimized conditions, the

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Figure 1. Hardware configuration and plumbing scheme.

+MS2 (931.70) CE (35): 10 MCA scans from Sample 6 (TuneSampleID) of RAP200ppb-InIsPs... Max. 6.9e4 cps.

700 720 740 760 780 800 820 840 860 880 900 920 940m/z, amu

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

6.9e4 864.6

814.6846.4832.5 882.5

931.7

706.4 828.6796.3 838.9 861.0758.8 898.3740.4 767.2710.3 820.9 949.0928.0911.4884.1842.6

Sirolimus 200 ppb by Inf-MSMS MCA-10 scans

+Q1: 0.100 to 0.852 min from Sample 1 (TuneSampleID) of Ju27-SIR-Ms.wiff (Turbo Spray) Max. 2.0e5 cps.

922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941m/z, amu

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

Intensity,

cps

931.6

932.6

926.9

933.6936.6

937.6

929.4934.5

938.4

935.6 939.6 940.3922.7 923.5 924.6

Sirolimus 200 ppb by Inf-MS

Sirolimus (Rapamycin) MS and MSMS spectraSirolimus (Rapamycin) MS and MSMS spectra

Figure 2. Sirolimus MS and MS/MS spectra. MS spectrum is zoomed on the [M+NH4]+ region.

Figure 3. Typical tracing obtained with the proposed hardware set-up on a blood sample.

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total run time was 2.5 minutes persample.

Figures 4 and 5 illustrate some of theoptimization steps, including theflowrate through the POROS®

Perfusion column (first dimension -Fig. 4), and the heating of the second-chromatography column (Fig. 5).

As proof of robustness, Figure 6 showsa comparison of traces obtained after a

significant number of injections usingthe proposed method with tracesobtained using a classical single-dimen-sion chromatography methodology. In the latter, the fluctuating baselinecan be ascribed to a non-exhaustivecleaning of the chromatographiccolumn from the sample matrix aftereach injection. Beyond some immediateinterferences like quenching effect onthe actual MRM reading, some otherinterferences are appearing after severalinjections. Here, accumulated samplematrix bulk starts to exit the columnbecause the latter overloaded.

The proposed methodology is effectivein removing, thanks to the POROS®

column trapping mechanism, the partof matrix characterized by high-molecu-lar compounds (namely proteins)making the instrumental set-up work

for unusual number of samples (so far,1000 injections) without any significantperformance degradation.

Other results further support theperformance of the proposed method:

• Figure 7 illustrates the linearity

obtained on calibrator samples.

• Figure 8 shows the retention-timereproducibility of the two-dimension

process by overlapping the traces forEverolimus obtained during the

calibration.

• Figure 9 documents an inter-dayreproducibility test over Cyclosporin A.

• Figure 10 shows the correlationobtained when comparing the results

of the proposed method with those ofan Immunoassay test.

Max. 6007.5 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5109 218 327 436 545 654 763

Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

9000

9500

9914

XIC of +MRM (5 pairs): 1219.0/425.5 amu from Sample 1 (Mix16160-Por2) of Mix16160-Por2-...

2.70

Effect of the Flowrate through the POROS Perfusion column on theEffect of the Flowrate through the POROS Perfusion column on therecovery (first dimension chromatography)recovery (first dimension chromatography):: column flowed @column flowed @

1.0, 1.0, 2.0, 2.0, and 2.5 mL/min. and 2.5 mL/min.

Max. 6188.8 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5109 218 327 436 545 654 763

Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

9000

9500

9921

2.70

XIC of +MRM (5 pairs): 1219.0/425.5 amu from Sample 1 (Mix16160-Por1) of Mix16160-Por1-... Max. 5705.0 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0109 218 327 436 545 654 763 870

Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

8880

Intensity,

cps

2.69

Figure 4. Effect of flowrate through the POROS® Perfusion column on the recovery (first dimension chromatography):results shown at 1.0 mL/min, 2.0 mL/min and 2.5 mL/min

Effect of the Temperature on the performances of a stressed analyticalEffect of the Temperature on the performances of a stressed analyticalcolumn (second dimension chromatography):column (second dimension chromatography):oven @ 20 d.C, oven @ 20 d.C, and @ 60 d.C. and @ 60 d.C.

Max. 1.0e4 cps.

7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.01397 1433 1468 1504 1540 1576 1612 1648 1683 1719 1755 1791

Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

80008282 9.78

8.99

8.22

XIC of +MRM (1 pair): 1203.0/425.5 amu from Sample 1 (TuneSampleID) of Csa-cold-warm.wiff... Max. 1.0e4 cps.

0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.889 107 125 143 161 179 197 215 233 251 269 287 305 322 340 358 376 394 412 430 448 466 484 501

Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

Intensity,

cps

0.78

1.53

2.31

2.36

Figure 5. Effect of temperature on the performances of a stressed analytical column (second dimension chromatogra-phy): oven at 20°C and at 60°C.

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Conclusion

The method presented in this paperproved to be fast (2.5 minute sample-to-sample cycle) and robust (more than

1,000 sample injections with no evidentcolumn saturation) without compro-mising the precision and accuracy ofthe assay. In addition, programmed

operation of the LC peripheralmodules using the ANALYST®

software reduced solvent consumptionby precisely controlling the high-flowrate regime during the sample-clean-upphase of the first dimensionchromatography.

References

1. B. Casetta, U. Ceglarek, J. Lembcke,J. Thiery, Poster Th-C 214 at 16thInternational Mass SpectrometryConference.

2. T. Koal, M. Deters, K. Resch, and V. Kaever, Poster TDM-I 58 atInternational Congress of TherapeuticDrug Monitoring and ClinicalToxicology-Basel 2003.

3. U. Ceglarek, J. Lembcke, G.M. Fiedler, M. Werner, J. Thiery,

Poster TDM-I 55 at InternationalCongress of Therapeutic

DrugMonitoring and ClinicalToxicology-Basel 2003.

4. B. Casetta, T. Koal and V. Kaever,Applied Biosystems Seminar atInternational Congress of Therapeutic

Drug Monitoring and ClinicalToxicology-Basel 2003.

5. T.Koal, M.Dieters, B.Casetta,

V.Kaever, Journal of ChromatographyB, 805 (2004) 215-222.

Figure 6. Comparison of the traces obtained on a blood sample with a previous method and with the proposed method.

Figure 7. Calibration curves with blood calibrators

BloodTacFinal.rdb (Tac): "Linear" Regression ("No" weighting): y = 0.0223 x + 0.153 (r = 0.9998)

0 5 10 15 20 25 30 35 40 45 50Analyte Conc. / IS Conc.

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0.85

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.351.37

BloodTacFull.rdb (CsA): "Linear" Regression ("No" weighting): y = 0.00193 x + 0.0435 (r = 0.9997)

0 100 200 300 400 500 600 700 800 900 1000Analyte Conc. / IS Conc.

0.0

0.1

0.1

0.2

0.2

0.3

0.3

0.4

0.4

0.5

0.5

0.6

0.6

0.7

0.7

0.8

0.8

0.9

0.9

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1.1

1.1

1.2

1.2

1.3

1.3

1.4

1.4

1.5

1.5

1.6

1.6

1.7

1.7

1.8

1.8

1.9

1.9

2.0

2.0

2.12.1

BloodSirResults.rdb (Sir): "Linear" Regression ("No" weighting): y = 0.0109 x + 0.0123 (r = 0.9996)

0 5 10 15 20 25 30 35 40 45 50 55Analyte Conc. / IS Conc.

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.32

0.34

0.36

0.38

0.40

0.42

0.44

0.46

0.48

0.50

0.52

0.54

0.56

0.58

0.60

0.62

0.64

0.66

TACRILOMUSfrom 1.96 to 52.9 ng/mL r=0.9998

SIROLIMUSfrom 2.17 to 58.7 ng/mL r=0.9996

CYCLOSPORIN Afrom 38.3 to 1035 ng/mL r=0.9997

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Acknowledgements

Applied Biosystems/MDS SCIEXacknowledges our collaborator, BrunoCasetta, and Therese Koal and VolkhardKaever from Medizinische HochschuleHannover, Germany, for providing themethod, samples and data for this appli-cation note.

Figure 8. EVEROLIMUS from blood Calibrators

Figure 9. Interday Reproducibility (n=3).

Figure 10. EMIT vs. LC/MS/MS

This research note describes customer-defined clinical research applications that have not been cleared or approved by the United States Food and Drug Administration or any otherregulatory agency for diagnostic or other clinical use. No certificate of conformity under the European IVD Directive has been obtained for the applications described in this note. Applied Biosystems does not promote or sell its instruments or other products for diagnostic or other clinical use. Applied Biosystems’ products are labeled “For research use only. Not for use in diagnostic procedures,” or are general laboratory instruments.

API 3000 is a trademark and ANALYST, LINAC and TurboIonSpray are registered trademarks of Applied Biosystems/MDS SCIEX, a joint venture between Applera Corporation and MDS Inc. All other trademarks are the sole property of their respective owners.

© 2005 Applera Corporation and MDS Inc. All rights reserved.

Printed in The Netherlands, 06/2005 Publication 114AP44-01

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