High Throughput Simultaneous Analysis of Immunosuppressants by ESI/ESI Dual Source Coaxial Flow Ion Source LC-MS/MS System
Sha Joshua Ye; Hui Qiao; Heather Gamble; John Tremblay
IONICS Mass Spectrometry, Bolton, Ontario, Canada

Introduction
LC-MS/MS is widely used to monitor immunosuppressive drug panels on a routine basis, for example fortherapeutic drug monitoring of organ transplant patients.[1] Multiplexing is a popular approach to speeding
the analysis time between individual samples. However, such techniques utilize a single electrosprayprobe and fast serial injections, typically requiring cumbersome wash cycles to ensure minimum
carryover.[2] This poster reports a novel approach to multiplexing the immunosuppressant analyses using
a single ion source equipped with two electrospray probes. Previous reports demonstrated linearity anduse of two ESI probes in a single source for analysis of Vitamin D [3]. Here we demonstrate fast quantitation of Sirolimus, Tacrolimus, Everolimus and Cyclosporin A using Ascomycin and Cyclosporin D as internal
standards.

ESI/ESI LC-MS Configuration
Eleuting PumpFor Analysis

AnalysisColumn 1ESIProbe 1

Autosampler
1Pretreatment
Column 1

ESI
Probe 2Eleuting Pump
For Analysis

AnalysisColumn 2

Autosampler
2Sample Introduction
Pump 1

Sample Introduction
Pump 2

Y = 1.807E-2x + 2.403E-30 Min

0.25 Min

1.5 Min

3.0 MinTime(min)
0.01
0.25
1.3
1.5
2.0
2.1
3.0

Solvent A Solvent B(%)(%)
1000100
0100
001000100
100
01000CY = 1.065E-2x + 3.416E-3
LC 1
Washing
Eleuting

Y = 1.399E-2x + 6.933E-3

DLC 20 Min

0.25 Min

3.0 Min

1.5 MinResultsEIC Chromatograms

Results: This method covers a concentration range of three orders of magnitude from 0.2
to 200 ng/mL for Tacrolimus, Sirolimus, Everolimus and 2 to 2000 ng/mL for Cyclosporin A,
while maintaining good linearity (R2 = 0.999) with 1/x weighting. The intraday and interday
variability for three levels QCs were all <7% and <11%, respectively. No interference orcross contamination was observed.
Figure 6: Intra and (Inter)-day Variability (%RSD)

Figure 4: EIC Chromatograms of Compounds for QC Level I in Whole Blood

Figure 1: LC Cycle TimeLoading Pump

BPretreatment
Column 2The total LC cycle time for an injection is 3 min and the sample analysis time is 1.5 min with alternating
injections from each LC. All the solvents used in this method are HPLC grade.
ElutingPump
Solvent B(%)
100
100
100
100
100
100
100

A

Y = 3.714E-2x + 2.414E-2

Figure 3: Injection Sequence

LC-MS/MS Conditions: The LC-MS/MS was performed using an IONICS 3Q 120 triple quadrupole mass
spectrometer (Bolton, ON Canada) with a Shimadzu UFLC system. 20 µL of supernatant were loaded on aporous R1/20 pretreatment column (30x2.1mm) for on-line washing with water for 0.25 minutes at a liquidflow rate of 3 mL/min, then eluted by an Imtakt Cadenza CD-C18HT analytical column (50x2.0mm, 3µm) atflow rate of 0.6 mL/min using Solvent A (water:methanol = 98:2, v/v, with 0.1% formic acid and 10mM ammonium acetate) and Solvent B (water:methanol = 2:98, v/v, with 0.1% formic acid and 10mM ammoniumacetate)
Mass Spectrometry Conditions: Electrospray Voltage: 5000V, HSID: 150°C, Nebulizer Gas: 450, Drying
Gas: 120, Heating Gas: 350, Source Temperature: 325°C.

Figure 5A: Calibration Curve for Sirolimus (0.2-200ng/mL) 5B: Tacrolimus (0.2-200ng/mL)
5C: Everolimus (0.2-200ng/mL) 5D: Cyclosporin (2-2000ng/mL)

Figure 2: The LC-MS/MS Setup Used During the Development of this Method.

MethodSample and Preparation: The Tacrolimus, Sirolimus, Everolimus and Cyclosporin A standard stock
solutions in liquid form were purchased from Cerilliant Inc (Round Rock, Texas) and stored at -4°C. Whole
blood spiked samples were cleaned up by mixing one volume of serum with two volumes 0.1 M ZnSO4
precipitation solution containing the internal standards: Cyclosporin D (1234/1217) and Ascomycin(809.6/756.6). Three level QCs were purchased from UTAK (Valencia, CA). The mixture was vortexed forone minute followed by centrifugation for 15 min. The supernatant was transferred to a clean vial for
quantitation.

Calibration Curves

Name

MRM

QC 1

QC 2

QC 3

Tacrolimus

821.6/768.5

3.5 (9.4)

1.4 (2.0)

2.1 (0.8)

Sirolimus

931.6/864.5

5.6 (4.9)

5.2 (2.1)

3.0 (2.0)

Everolimus976.7/908.7

6.6 (10.1) 3.1 (2.1)

3.5 (0.9)

Cyclosporin A

1220.0/1203

1.9 (7.5)

3.5 (1.0)

3.6 (1.9)

Conclusion

Valve
Position
Washing
ElutingSignal to Noise Ratio (S/N)

Washing

A sensitive, reliable and accurate LC-MS/MS method was developed and validated forquantification of Tacrolimus, Sirolimus, Everolimus and Cyclosporin A in whole blood. The
use of an ESI/ESI novel dual source allows a sample analysis time of only 1.5 minutes,
which doubles the throughput. This LC-MS/MS method requires simple samplepreparation and is well-suited for routine therapeutic drug monitoring ofimmunosuppressive drugs.
Reference: [1] Christoph Seger1, et al. A rapid HPLC-MS/MS method for the simultaneous quantification of cyclosporine A,tacrolimus, sirolimus and everolimus in human blood samples. Nature Protocols, Vol 4, 526-534 (2009)[2] Vogeser M, Spöhrer U. Pitfall in the high-throughput quantification of whole blood cyclosporin A using liquid chromatography-tandem mass spectrometry. Clin Chem Lab Med. 43(4):400-402 (2005).
[3] Sha Joshua Ye, et al, Method Development Time Reduction In Clinical Applications By Multiplexing Two HPLC Analyses
Using a Dual Coaxial Flow Ion Source , 2011 ASMS meeting poster

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www.ionics.ca*Methods Described in this Poster are for Research use only