automated spe for capillary microsampling poster
TRANSCRIPT
IntroductionAlternatives to animal testing and the development of methods that helpreducing the number of animals in preclinical studies are much soughtafter. In capillary micro sampling, a low volume of blood (for example 30µL) is collected in a capillary, centrifuged and an exact volume of plasma(for example 10 µL) transferred into another capillary. For analysis, theplasma is washed or blown out into a sample tube and diluted to avolume, that can be handled reliably and reproducibly. In contrast toDBS techniques, plasma is processed and analyzed, thus avoidinginvestigation of critical parameters like effect of hematrocit and offeringstandard procedures for addition of stabilizers or internal standardsduring analysis. Even though necessary, dilution should be minimized asit directly affects the achievable limits of quantification. In this poster wedescribe an automated SPE procedure using small, single usecartridges in a modified CTC liquid handling auto sampler. Results fromthis approach are compared to results obtained from identical samplesprocessed manually by protein precipitation.
Evaluation of an automated SPE method for samples derived from capillary microsampling
Paul-Gerhard Lassahn1, Petra Lombardi1, Winfried Wagner-Redeker1, Marie-Claude Roubaudi-Fraschini2, Reto Bolliger3, Guenter Boehm3
1 Swiss BioAnalytics AG, Basel, Switzerland2 Debiopharm International S.A., Lausanne, Switzerland
3 CTC Analytics AG, Zwingen, Switzerland
ExperimentalGeneral: Samples were processed by dilution of spiked plasma with water containing the internal standardfollowed by online sample-preparation described below.Working-solutions and sample preparation: The analyte (MW 581) was dissolved in DMSO and diluted inACN/DMSO (50:50, v/v) to obtain working-solutions at concentrations twenty times higher than those of thefinal CAL and QC samples. Aliquots of the working-solutions were spiked into blank rat plasma and diluted withwater containing the internal standard in a ratio of 1:10 (v/v). The samples were vortexed, centrifuged andtransferred to the ITSP sample-preparation plate.Online-sample-preparation: ITSP (ITSP Solutions Inc.) cartridges were conditioned with 80µL MeOH and 80µL0.2% aqueous formic acid before loading 10µL sample. After drying the cartridge with 80µL air, the ITSPcartridges were washed twice with 80µl 0.2% aqueous formic acid and dried again. The compounds wereeluted using 80µL EtOH and 10µL of the eluate was injected onto the analytical column. Sample-preparation ofeach plasma sample was performed directly before the injection (see Figure 1 for the workflow and the resultsof the optimization process).Chromatography: 10 µL of the extracted samples were injected onto a Thermo Hypersil Gold (2.1 x 100 mm, 3µm) analytical column. Eluent A consisted of 0.5% aqueous formic acid, eluent B of 0.5% formic acid in ACNand LC flow rate was 0.5 mL/min. The eluent composition was changed linearly from 70% to 5% A in 2.25minutes. Eluent composition was kept at 5% B for one minute and then went back ballistically to startingconditions.Calibration range: 50.0 - 25’000 ng/mL (high-range, HR) and 5.00 - 2’500 ng/mL (low-range, LR).Instrumentation and MS-method: TSQ Quantum Ultra (Thermo Scientific), Rheos 2200 quarternary pump (FluxInstruments), HTX PAL autosampler (CTC Analytics).Data-Processing: Raw data were acquired with Xcalibur 2.0 software and processed with LCQuan 2.5 (ThermoScientific).Validation parameters: Intra- and inter-assay accuracy and precision, linearity, and carry-over.
Table 1. MS-parameter (SRM-assay).
www.swissbioanalytics.com
Results
Blank rat plasma was spiked with the analyte and diluted with watercontaining the internal standard in a ratio of 1:10 (v/v). CALs and QCswere prepared to cover concentrations of 50.0 - 25’000 ng/mL in thehigh-calibration range and 5.00 - 2’500 ng/mL in low calibration range.The diluted plasma samples were transferred to the ITSP sample-preparation plate and automatically extracted directly before analysis.Sample preparation and LC-MS/MS measurements were performed inparallel using the “Prep Ahead Function” of the CTC autosampler(Figure 4). Chromatograms of the lowest calibration standard Cal1 arepresented in Figure 2 and compared with the chromatograms of thesame calibration standard prepared by protein-precipitation.Three independent precision/accuracy runs were conducted for bothcalibration ranges and the intra- and inter-run variabilities arepresented in Table 2. An example of a typical calibration curve foreach range is presented in Figure 3. Both assays gave very similarresults and the intra- and inter-run performance is equivalent to thedata obtained using a standard protein-precipitation method.The carry-over in the analytical assay does not meet the acceptancecriteria of the EMA guideline but can be significantly reduced by using
Analyte Parent ion mass [Da]
Product ion mass [Da]
Collision energy [V]
Analyte 582.3 255.1 35
Internal Standard 588.3 487.2 30
ESI positive, spray voltage 3.5 kV, capillary temperature 350 °C, collision gas pressure (Ar) 1.5 mTorr, scan-time 100 msec
Figure 6. Linearity of the loading-volume and capacity of cartridges.
Loading
Eluting
Area Analyte Area IS Area RatioArea Analyte Area IS Area Ratio
5µL 10µL Injection volume 40µL
Conclusions
•The automated workflow described above gave equivalentresults (LLOQ, precision, accuracy) compared to samplesprocessed manually by protein precipitation. The overalltime/sample is slightly higher for the automated protocol.However, since only minimal intervention is required, processsafety is superior and human resources are freed up forother tasks.•The automated protocol may be further optimized in severalaspects. As shown in Figure 7, a volume of 20 µL is sufficientfor complete elution of the analyte from the cartridge. Figure6 indicates that an loading volume of 2-5 µL diluted plasma issufficient allowing the use of low-volume samples. Bothaspects are important for further optimizing this protocol foranalysis of samples originating from studies usingmicrosampling techniques.•Methods for the automated optimization of the SPEconditions exist and will be applied in future studies.
Reference: Nilsson LB, Ahnoff M, Jonsson O; Bioanalysis(2013) 5(6), 731–738
Low-Range
Y = 0.000781592+0.00128746*X+1.0801e-008*X^2
R^2 = 0.9990 W: 1/X
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Figure 3. Calibration-curves (low-range assay left, high-range assay right).
Figure 1. Workflow and results of the optimization process.
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Elution pre-testing:(a) 10mM NH4Fo pH9 (b) 10mM NH4Fo pH9/MeOH (50:50) (c) 10mM NH4Fo pH9/ACN (50:50)(d) ACN (e) MeOH(f) ACN/MeOH (50:50) (g) ACN/MeOH (75:25) (h) ACN/MeOH (75:25) (i) EtOH
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Washing variable Washing variable
Figure 2. Comparison of protein-precipitation (PP) and ITSP at LLOQ.
J:\Projekte\...\Cal1_LR_Precipitation_02 8/23/2013 1:40:16 PM 9 3 13020 CAL1 1 1
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TIC F: + c ESI SRM ms2 [email protected] [255.050-255.150] MS Cal1_LR_Precipitation_02
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TIC F: + c ESI SRM ms2 [email protected] [487.150-487.250] MS Cal1_LR_Precipitation_02
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TIC F: + c ESI SRM ms2 [email protected] [255.050-255.150] MS 04112013_05
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TIC F: + c ESI SRM ms2 [email protected] [487.150-487.250] MS 04112013_05
J:\Projekte\...\Cal1_HR_Precipitation 7/30/2013 12:10:33 PM 2 3 13020 CAL1 1 1
RT: 0.0 - 4.0 SM: 7G
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TIC F: + c ESI SRM ms2 [email protected] [255.050-255.150] MS Cal1_HR_Precipitation
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TIC F: + c ESI SRM ms2 [email protected] [487.150-487.250] MS Cal1_HR_Precipitation
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TIC F: + c ESI SRM ms2 [email protected] [255.050-255.150] MS 24102013_5
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TIC F: + c ESI SRM ms2 [email protected] [487.150-487.250] MS 24102013_5
Low-Range
Analyte
InternalStandard
PP ITSPHigh-Range
PP ITSP
0.0 100.0 200.0 300.0 400.0 500.0 600.0
Zentrifugieren
Verdünnen
Transfer ITSP
Fällung IS
MS
0.0 200.0 400.0 600.0 800.0 1000.0 1200.0 1400.0
PP
PPITSP
PPITSPG
rad
ien
t
5min
7min
10min
Run-Time [min]
ITSP# of Samples: 100
centrifugediluteTransfer ITSP-PlateProtein precipitationMS-measurement
# o
f S
am
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s
Run-Time [min]
100
200
400
Sample-Preparation:
Figure 5. Comparison of protein-precipitation (PP) and ITSP (#Samples: variable; gradient-time: variable; ITSP: sample-preparation7min in parallel; PP: pre-injection-time 1.0min).
PPITSP
PPITSP
PPITSP
at LLOQ (LR) at LLOQ (HR) > LLOQ (LR) > LLOQ (HR)
PrecIntra 9.7-10.5% 5.0-6.0% 1.5-10.5% 1.4-8.3%
AccIntra 93.2-107.4% 95.7-113.0% 101.0-105.2% 95.5-113.5%
PrecInter 11.1% 8.9% 4.4-9.5% 2.5-8.1%
AccInter 100.6% 105.1% 101.0-105.2% 104.8-108.6%
Table 2. Intra- and inter-run precision and accuracy (3 runs, [%]).
Calibration-Range Carry-over*
[% of CAL8]
Carry-over*
[% of Cal1]
Low-Range [1st Blank after Cal8] 0.6 ** 229.7 **
Low-Range [2nd Blank after Cal8] 0.2 ** 80.3 **
High-Range [1st Blank after Cal8] 0.7 278.6
High-Range [2nd Blank after Cal8] 0.3 108.5
* Standard-Wash-solution; Wash 1: H2O/EtOH (50:50, v/v); Wash 2 ACN/MeOH/i-Prop (1:1:1, v/v/v)
** Carry-Over can be significantly reduced using 0.3% Tween in Wash-solution 1 (below 30% of Cal1 for the 2nd Blank after Cal8)
Table 3. Carry-over.
Figure 4. Prep Ahead Function of the CTC autosampler.
Area Analyte
20µL10µL 80µL
Area Analyte
40µL
Figure 7. Volume of the elution solvent EtOH (area of analyte normalized to concentration).
0.3% Tween as an additive in the aqueous wash-solution(see Table 3).The influence of the amount of (diluted) plasma loaded ontothe cartridge and the volume of the extraction solvent ispresented in Figures 6 and 7. Elution from the ITSPcartridge is complete with as little as 20 µL of extractionsolvent (Figure 7) resulting in a factor 4 higherconcentration of the analyte in the eluate compared to thestandard-elution method. The capacity of the cartridges hasbeen tested between 2 to 40 µL diluted plasma (Figure 6).The area ratio of analyte to internal standard is constantacross this range and demonstrate excellent performanceof the method across a wide range of sample volumes.The sample-preparation time and the overall run-time ofthe automated and manual method are compared in Figure5. Since only minimal manual intervention is required for theautomated approach, this workflow offers significantadvantages by high sample-workload and longer LC-MS/MS run-times.