Alternative Sample Preparation for Immunosuppressant Analysis Authors: D. Dosch(1), C. Mang (1, 3), C. Berchtold (2) and G. Schlotterbeck (2) (1) Hamilton Bonaduz AG, 7402 Bonaduz, Switzerland ; (2) Fachhochschule Nordwest Schweiz, 4058 Basel, Switzerland ; (3) Corresponding author:
[email protected]
Introduction Protein precipitation is the commonly used sample preparation technique to prepare Immunosuppressive drugs out of whole blood for LC-MS analysis. The removal of the precipitated proteins from blood is crucial and achieved by centrifugation at high g-forces. This process has successfully automated. Filtration also allows for the use of a positive pressure device instead of an integrated centrifuge avoiding transport steps of the filtration and collection plate into the centrifuge and the preparation of counterweights for the centrifuge. Positive pressure devices also allow for the implementation of SPE assays on the same platform without the need of changing hardware or additional cost.
Fig. 1: on top, the Hamilton MassSTAR workstation Fig. 2: [MPE]2 Positive Pressure, Extraction & Evaporation Module
Methods Sirolimus (SIR), Everolimus (EVE), Tacrolimus (TAC) and Cyclosporin A (CSA) were analysed by LC-MS using the automated CHROMSYSTEMS MassTox® Immunosuppressant kit using an Hamilton Microlab STARlet IVD System for sample extraction. Blank blood, calibrators and controls were prepared as shown in figure 3 to test for the applicability of positive pressure for the transfer of samples from the filter to the collection plate. The transfer of the samples from the filter plate to the collection Plate was assessed using either the centrifuge or the [MPE]2, the Hamilton’s positive pressure device. After the final incubation step, the filter plate was manually moved to an [MPE]2 device and stacked on top of a Hamilton 2ml DWP. The stack was loaded into the [MPE]2 and clamped against the manifold. Using the “Elution to Plate” function with 35psi for 3s and 10 psi for 30s all samples were transferred to the collection plate. For each sample an analysis was done using either the centrifuge or [MPE]2 for sample preparation. The sample analysis was performed on an Agilent 6410 triple quad mass spectrometer and an Agilent 1200 LC-System according to the Chromsystems protocol. The batches were prepared in duplicate for Centrifuge and [MPE]2.
Cross Contamination: The signal for all analyte in the Stress Blank Samples (SB) is clearly below the lower limit of quantification (LLOQ) (Fig. 5).
3
4
5
6
7
8
A
blank
1
2
3
4
5
6
blank
B
SB SB SB SB SB SB SB SB SB SB SB SB
C
SB
D
SB SB SB SB SB SB SB SB SB SB SB SB
E
SB SB SB SB SB SB SB SB SB SB SB SB
F
SB
G
SB SB SB SB SB SB SB SB SB SB SB SB
H
blank
SB
6
SB
1
2
6
6
3
SB
SB
4
6
6
5
SB
6
SB
6
10
SB SB
blank
12
C1 C2 C3 C4
SB SB
6
11
6
6
SB
CSA
6
9
EVE
2
SIR
Linearity: Standard curves were calculated using the calibrator levels 1 – 6 as supplied in the Chromsystems Calibrator XL set. For all standard curves the linearity was >0.98 which is comparable with the result obtained when the centrifuge was used for the filtration step (Fig. 4).
1
TAC
Results
SB
C1 C2 C3 C4
Fig. 3: Plate Setup for Sample Transfer Stress Test
BATCH
SIR
EVE
TAC
CSA
[MPE]² - 1
Y=0.349295*x + 0.008145 / R^2=0.98
Y=0.218601*x + 0.013621 / R^2=0.99
Y=0.150949*x + 0.039754 / R^2=0.99
Y=0.455158*x + 0.026868 / R^2=0.99
[MPE]² - 2
Y=0.374058*x + 0.032073 / R^2=0.99
Y=0.236324*x + 0.007243 / R^2=0.99
Y=0.164230*x + 0.070952 / R^2=0.99
Y=0.499757*x + 0.012128 / R^2=0.99
CENTRIFUGE -1
Y=0.372450*x + 0.005939 / R^2=0.99
Y=0.230890*x + 0.047686 / R^2=0.99
Y=0.176867*x +0.031693 / R^2=0.99
Y=0.528662*x + 0.016966 / R^2=0.99
CENTRIFUGE -2
Y=0.377034*x + 0.030557 / R^2=0.99
Y=0.233942*x + 0.027998 / R^2=0.99
Y=0.16752*x + 0.054604 / R^2=0.99
Y=0.496309*x + 0.013551 / R^2=0.99
Fig. 4: Standard Curve of the four analyte ([MPE]2) and linear regression comparison (centrifuge)
Discussion
Percent of LLOQ
EVERLOLIMUS
Percent of LLOQ
SIROLIMUS
40
60
40
20
[MPE]2 1
[MPE]2 2
Centrifuge 1 Centrifuge 2
100
[MPE]2 1
[MPE]2 2
Centrifuge 1 Centrifuge 2
[MPE]2 1
[MPE]2 2
Centrifuge 1 Centrifuge 2
100
80
60
40
20
[MPE]2 1
[MPE]2 2
Centrifuge 1 Centrifuge 2
80
Percent of LLOQ
CYCLOSPORIN A
With that the exchange of the centrifuge against an positive pressure device for filtration is possible which allows the usage of the same instrument also for standard SPE assays without a need for additional hardware.
60
80
20
Percent of LLOQ
Cross-Contamination can further be reduced using filter plates with nozzles reaching into the target well. The linearity of the standard curves are similar to those of the centrifuge and also relative response rate is equal indicating no sample loss.
100
80
TACROLIMUS
All cross-contamination values achieved in using Hamilton’s [MPE]2 positive pressure device are below the lower limit of quantitation of the Chromsystems kit which clearly indicates that the centrifuge can be exchanged by a positive pressure device without having a spray effect. However, the transfer parameters time and pressure have to be optimized for the specific application. It should be noted, that cross-contamination below the LLOQ was achieved using the default “flat-bottom” Filter plate.
100
60
40
20
Fig. 5: Box plots represent the amount of cross contamination observed using either the centrifuge or MPE2 for sample transfer
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