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3 Fully automated platform for determination of immunosuppressant drugs in whole blood The Immunosuppressant standards and the Internal were rstly analysed by ow injection to optimize mass spectrometer parameters. All …

Fully automated platform for determination of

immunosuppressant drugs in whole blood

Introduction

Therapeutic drug monitoring of four major

immunosuppressant drugs, Cyclosporine A, Tacrolimus,

Sirolimus and Everolimus, is well established. Overdosing

with these critical dose drugs can cause serious toxicity

and long term morbidity, while organ rejection can occur

if a patient is under dosed. Nowadays clinical laboratory

has two main choices in technologies: immunoassay or

chromatography based methods. LC–MS/MS's superior

specicity makes it the presumptive gold standard in

immunosuppressant quantitation. It relieves the method

from common interferences that plague immunoassays

such as metabolites that have structural resemblance and

interfering antibodies. However, current LC–MS/MS

platforms demand personnel expertise and tedious

sample preparation and sample throughput is generally

much lower compared to immunoassays. We report a

fully automated procedure for the quantitation of four

major immunosuppressant in whole blood samples,

increasing data quality/precision, throughput and safety

(The work described herein is for research use only).

Methods

The quantitative analysis of Immunosuppressant was

performed using reagents provided in Chromsystems

“MassTox

” ONEMinute Kit (ref, 93900). The

Immunosuppressant and Internal standard were

monitored using UHPLC-MS/MS system (Nexera X2 and

LCMS-8050, Shimadzu, Kyoto, Figure 1)).

Sample preparation was performed using Precipitation

reagent, Extraction buffer and Internal standard set.

Analytical performance of the method was monitored

using whole blood calibrators and whole blood QC.

Automatic sample preparation was performed using

CLAM-2000 module (Shimadzu, Kyoto) Figure 1.

Figure 1: CLAM-2000 online with Nexera X2 system and LCMS-8050 triple quadrupole mass spectrometer.

Fully automated platform for determination of

immunosuppressant drugs in whole blood

The Immunosuppressant standards and the Internal were rstly analysed by ow injection to optimize mass

spectrometer parameters. All compounds were detected as positive ion choosing the MRM transitions listed in Table 2.

Then Immunosuppressant standard mix was used to set-up chromatographic separation (Table 1).

LC-MS/MS analysis

Result and discussion

The CLAM-2000 was programmed to perform sample extraction and protein precipitation followed by ltration and

sample collection. The ltrated sample was then automatically transported using an arm from the CLAM-2000 to the

HPLC for LC-MS/MS analysis and no human intervention was required (Fig 2).

Fully Automated sample preparation

Column Temp. : 65 °C

Time Program : 0.3 min (trap load); 1.5 min (elution); 2.3 min (stop)

Injection Volume : 5 μL

[LC] NexeraX2 System

Ionization : ESI Positive

Nebulizer Gas : 3 L/min

Interface temperature : 300 °C

Desolvation Line : 250 °C

Heat Block temperature : 400 °C

Drying Gas : 10 L/min

Scan Type : MRM

[MS] LCMS-8050

Table 1: Analytical Condition

Table 2: MRM Transitions

Cyclosporin A

Tacrolimus

Sirolimus

Everolimus

Cyclosporin A-d

Everolimus-d

Sirolimus-d

Tacolimus-

Compound

1219.90 > 1202.80

821.60 > 768.30

931.70 > 864.50

975.70 > 908.50

1231.90 > 1214.80

979.60 > 912.40

934.60 > 864.40

824.60 > 771.40

MRM transition

Fully automated platform for determination of

immunosuppressant drugs in whole blood

Figure 3: Linearity and LLOQ.

Figure 2: CLAM2000 fully automated sample preparation and analysis.

Due to the overlapped sample preparation the throughput of the instrument was

1 result each 3.7 minutes for Immunosuppressant quantication.

Linearity and Accuracy were evaluated using reference

whole blood calibrators (7 levels) spanning from a wide

range of concentrations (Cyclosporin 16.5 – 1100 µg/mL,

Everolimus 0.86 – 32.7 µg/mL , Sirolimus 0.9 – 32.7

µg/mL, Tacrolimus 0.96 – 34.8 µg/mL). For all analytes

Linearity was >0.99 (see Table 3) with S/N >25 for LLOQ

levels (Figure 3). Precision of the assay was evaluated

using Chromsystems reference materials (whole blood

controls levels) spanning from low concentration to

highest concentration for each analyte (Table 4).

Linearity, Accuracy and Precision

Reagent

Dispensing

Extr. Buffer

50 ul

Sample

Dispensing

Whole blood

25 ul

Internal STD

12.5 ul

Reagent

Dispensing

Stirring

2200 rpm

Incubation

Room

temperature

Reagent

Dispensing

Filtration

Aspiration

Direct UHPLC

connection for

LCMS analysis

(2.3 min)

Stirring

2200 rpm

Parallel/Sequential sample preparation and analysis

Reagent

Dispensing

MetOH

20 ul

Sample 3

processing

Sample 2

processing

Sample 1

result

Prec. Reagent

125 ul

Table 3: Linearity & Accuracy.

Cyclosporin A

Tacrolimus

Sirolimus

Everolimus

Compound

90.4%-103.6%

99.7%-101-3%

95.1%-105.5%

96.9%-107%

Accuracy

0.998

0.997

0.998

0 500 1000 Conc. Ratio

0,0

2,5

5,0

7,5

Area Ratio

1,5 2,0

5000

10000

15000

0 10 20 30 Conc. Ratio

0,0

0,5

1,0

1,5

2,0

2,5

Area Ratio

1,50 1,75

250

500

750

1000

1250

1500

0,0 25,0 Conc. Ratio

Area Ratio

1,50 1,75

500

1000

1500

2000

0 10 20 Conc. Ratio

0,0

0,5

1,0

1,5

2,0

Area Ratio

1,5 2,0

2500

5000

7500

10000

Cyclosporin A Everolimus Sirolimus Tacrolimus